Type II protein kinase A regulates CFTR in airway, pancreatic,
and intestinal cells
Wendy K.
Steagall1,
Thomas J.
Kelley1,
Rebecca J.
Marsick1, and
Mitchell L.
Drumm1,2
Departments of
1 Pediatrics and
2 Genetics and Center for Human
Genetics, Case Western Reserve University, Cleveland, Ohio
44106-4948
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ABSTRACT |
The type of
protein kinase A (PKA) responsible for cystic fibrosis transmembrane
conductance regulator (CFTR) activation was determined with adenosine
3',5'-cyclic monophosphate analogs capable of selectively
activating type I or type II PKA. The type II-selective pair stimulated
chloride efflux in airway, pancreatic, and colonic epithelial cells;
the type I-selective pair only stimulated a calcium-dependent efflux in
airway cells. The type II-selective analogs activated larger increases
in CFTR-mediated current than did the type I-selective analogs.
Measurement of soluble PKA activity demonstrated similar levels
stimulated by type I- and type II-selective analogs, creating an
apparent paradox regarding PKA activity and current generated. Also,
addition of forskolin after the type I-selective analogs resulted in an
increase in current; little increase was seen after the type
II-selective analogs. Measurement of insoluble PKA activity stimulated
by the analogs resolved this paradox. Type II-selective analogs
stimulated three times as much insoluble PKA activity as the type
I-selective pair, indicating that differential activation of PKA in
cellular compartments is important in CFTR regulation.
cystic fibrosis; adenosine 3',5'-cyclic monophosphate; ion channels; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) is an adenosine 3',5'-cyclic monophosphate
(cAMP)-regulated chloride channel activated by phosphorylation by
protein kinase A (PKA). CFTR is expressed in a variety of tissues,
including respiratory epithelium, sweat and pancreatic glands, and
intestinal epithelial cells. Mutations in CFTR may lead to cystic
fibrosis, a recessive autosomal lethal disease characterized by
progressive respiratory failure and infection, exocrine pancreatic
dysfunction, gastrointestinal blockages, high sweat chloride
concentrations, and infertility (29). Regulation of CFTR-mediated
chloride permeability is achieved through two events: phosphorylation
of multiple serines in the R, or regulatory, domain of the protein,
followed by binding and hydrolysis of ATP at the nucleotide-binding
folds (5, 12, 14). It has been shown that some mutant CFTR channels can
be activated pharmacologically by compounds that stimulate or inhibit components of the PKA pathway such as
-adrenergic receptors, adenylate cyclase, phosphodiesterases, phosphatases, and PKA itself (3,
10, 15, 16). The existence of several isoforms of each component,
however, adds complexity to determining which pharmacological agent
could stimulate or inhibit this pathway. To more completely understand
the regulation of CFTR and to develop more specific methods of
pharmacological activation of CFTR mutants, we characterized the type
of PKA involved in CFTR regulation in cell lines derived from three of
the tissues most clinically affected in cystic fibrosis: airway,
pancreas, and intestines.
PKA is a holoenzyme consisting of a regulatory subunit dimer and two
catalytic subunits. Each regulatory subunit contains two sites for cAMP
binding; each site has different affinities for cAMP and acts
cooperatively to bind cAMP. On binding of four molecules of cAMP, the
affinity of the regulatory subunits for the catalytic subunits
decreases 10,000- to 100,000-fold, and the catalytic subunits are
released and may phosphorylate specific protein targets (reviewed in
Refs. 11 and 27). PKA exists as two types, I and II, which are defined
by the type of regulatory subunit present in the holoenzyme, RI and
RII, respectively. The regulatory subunits differ in molecular weight,
protein sequence, phosphorylation state, tissue distribution,
subcellular location, and other biochemical properties (11, 27). The
RII subunits have been found to be associated with A kinase-anchoring
proteins (AKAPs), which may sequester the type II PKA in specific
cellular compartments (7, 20). The localization of types may serve to
enhance the specificity of the PKA reaction by compartmentalization of
PKA with preferred substrates. Isoforms of each of the regulatory subunits have been found (RI
, RI
, RII
, RII
), with the
-isoforms being expressed in most cells; there are also three types
of catalytic subunits (C
, C
, and C
). The catalytic subunits
are found interchangeably with the regulatory homodimers (11, 27).
To better understand the activation pathway of CFTR, we designed
experiments to determine which type of PKA regulates CFTR in airway,
pancreatic, and intestinal cells. cAMP analogs that preferentially
activate a specific type of PKA were used in assays designed to measure
CFTR-mediated chloride secretion. These cAMP analogs not only have high
stability and membrane permeability, they also have selective binding
affinities for the A or B cAMP-binding sites of the regulatory subunit
isoforms (RI or RII) (reviewed in Ref. 4). By combining pairs of
analogs that are selective for A and B sites of a particular PKA type,
one can preferentially activate that type and measure a specific
outcome of PKA activation. Rat parotid gland secretion (13, 23),
inhibition of T lymphocyte proliferation (26), steroidogenesis in rat
adrenal (30) and Leydig cells (21), and growth inhibition of
transformed cancer cells (6, 24) are just some of the phenomena that
have been examined through the use of site-selective analogs to
determine the type of PKA involved in each process. It is hoped that,
by identifying the type of PKA responsible for CFTR regulation,
pharmacological tools to activate mutant CFTRs can be developed.
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MATERIALS AND METHODS |
Drugs.
8-(6-Aminohexyl)aminoadenosine 3',5'-cyclic monophosphate
(AHA-cAMP), 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic
monophosphate (CPT-cAMP),
N6-benzoyladenosine
3',5'-cyclic monophosphate
(N6-benzoyl-cAMP),
forskolin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
(DIDS), glibenclamide, and bumetanide were obtained from Sigma
Chemical, 8-piperidinoadenosine 3',5'-cyclic monophosphate (PIP-cAMP) from BioLogs, and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and nystatin from Calbiochem.
Cell culture.
Calu-3 cells (25) were grown in Eagle's minimal essential medium (MEM)
with Earle's balanced salt solution supplemented with sodium pyruvate,
nonessential amino acids, 10% fetal bovine serum, and
L-glutamate; T84 cells (1) in a
1:1 mix of Ham's F-12 and Dulbecco's minimal essential medium (DMEM)
plus 5% fetal bovine serum and
L-glutamate; and Capan-1 cells
(2, 17) in RPMI 1640 medium, 15% fetal bovine serum, and
L-glutamate; all were aerated
with 5% CO2 at 37°C. All cell
lines were obtained from the American Type Culture Collection
(Rockville, MD). Cells were seeded at a density of 1-5 × 105
cells/cm2. For isotopic efflux
assays, cells were grown to confluency in six-well plates; cells for
Ussing chamber experiments were grown to confluency on collagen-coated
Millicell-CM permeable supports (0.63 cm2) from Millipore. Capan-1 and
T84 cells were cultured on plastic before PKA activity measurements.
Isotopic efflux assays.
The chloride efflux assay is a modification of that described by
Venglarik et al. (28), in which chloride is substituted for iodide.
Cells were incubated for 1.5 h in
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered Ringer solution (HBR) consisting of (in mM) 10 HEPES (pH 7.4), 138 NaCl, 5 KCl, 2.5 Na2HPO4,
1.8 CaCl2, 1 MgSO4, and 10 glucose containing 5 µCi of Na36Cl (Amersham). Cells
were washed four times with HBR to remove extracellular
36Cl, and then at 30-s intervals
the buffer in which the cells were incubated was removed, transferred
to a scintillation vial, and replaced with fresh HBR. cAMP analogs (50 µM each) were added at 180 s and were present until 480 s, when
agonist-free HBR was again used. After the final efflux interval, cells
were lysed with 1% Triton X-100 in HBR for 20 min. Scintillation fluid
was added, and vials were counted in a Beckman LS 5801 liquid
scintillation counter. When BAPTA-AM (10 µM) was used, the cells were
incubated for 30 min with BAPTA-AM before efflux with the analogs.
Efflux was calculated as the percentage of
36Cl secreted into the HBR since
the previous time point
{[cpmx
cpm(x + 1)]/cpmx,
where x and x + 1 represent successive time
points} and plotted as a function of time. Quantitation of
responses was expressed as the average of the rates at each time point
during stimulation with drug.
Short-circuit current measurements.
Short-circuit current
(Isc) was
measured on monolayers of cells grown on permeable supports, seeded at
~8 × 105
cells/cm2, and used 7-14 days
after seeding. Cells were mounted in a modified Ussing chamber (WPI)
with bicarbonate-buffered Ringer solution (KBR) consisting of (in mM)
25 NaHCO3, 115 NaCl, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, 1 MgSO4, and 10 glucose (pH 7.4) on
the basolateral side and a chloride-free Ringer solution consisting of
(in mM) 115 sodium cyclamate, 25 NaHCO3, 5 potassium gluconate, 2.5 Na2HPO4, 3.6 calcium cyclamate, 1.0 MgSO4,
and 10 glucose (pH 7.4) on the apical side. All experiments were
carried out at 37°C, and KBR was continuously bubbled with 95%
O2-5%
CO2 by air-lift circulators. Voltage clamping was accomplished with a voltage-current clamp (model
DVC 1000, WPI, Sarasota, FL). Transepithelial voltage
(Vt) was
recorded through agar bridges (4% agar in KBR) connected to balanced
calomel electrodes.
Vt was clamped to
0, and Isc was recorded through Ag-AgCl electrodes. Transepithelial resistance was
calculated by measuring the change in
Isc
(
Isc) in
response to a +2-mV clamp of
Vt (3-s pulse
every 30 or 40 s) and was corrected for solution resistance. Data were
collected on a MacLab/4e (AD Instruments, Milford, MA). In experiments
where the basolateral membrane was permeabilized, nystatin
(0.4-0.6 mg/ml) was added to the basolateral side 10-30 min
before the addition of type II-selective cAMP analogs. Average
resistances across cell monolayers were 288, 66, and 102
· cm2 for
Capan-1, T84, and Calu-3 cells, respectively.
Assay for activated PKA.
Capan-1 or T84 cells were grown to confluency on plastic dishes and
then assayed. Cells were incubated at 37°C for 15 min with AHA-cAMP
plus PIP-cAMP or CPT-cAMP plus
N6-benzoyl-cAMP
(50 µM each analog), then washed, lysed in extraction buffer [5
mM EDTA, 50 mM tris(hydroxymethyl)aminomethane, pH 7.5], and spun
for 5 min at 14,000 g in a microfuge.
The supernatant was kept as the soluble fraction, and the pellet was
resuspended in extraction buffer plus 1% Triton X-100 and designated
the particulate fraction. PKA activity was measured according to the
manufacturer's directions (PKA assay system, GIBCO BRL). Briefly, for
each condition, four reactions were incubated for 5 min at 25°C
with an oligopeptide (kemptide) as the substrate. Activity of the
lysate less activity of the lysate in the presence of the PKA inhibitor
gave the PKA activity of the fraction. Activity of the lysate in the
presence of 4 µM cAMP less activity in 4 µM cAMP plus the PKA
inhibitor gave the total PKA activity capable of being stimulated in
that fraction of the cell. Pilot experiments were carried out to
determine the optimal amount of lysate necessary to keep the rate of
kemptide phosphorylation in the linear range. The amount of PKA
activated by the analogs was calculated as the percentage of PKA
activated relative to the total cAMP-activatable PKA activity in that
fraction.
Statistics.
Paired comparisons were evaluated by
t-test. Significance was arbitrarily
chosen as
= 0.05.
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RESULTS |
We investigated which type of PKA activated CFTR in cell lines used as
models for three tissues affected in cystic fibrosis: Capan-1, a human
pancreatic adenocarcinoma cell line; T84, a human colon carcinoma cell
line; and Calu-3, a human airway epithelial cell line. CFTR function
was measured after incubation of the cells with combinations of cAMP
analogs selective for type I or type II PKA. The combination of
AHA-cAMP and PIP-cAMP was used to preferentially activate type I PKA,
inasmuch as AHA-cAMP is selective for the B site of RI, whereas
PIP-cAMP selectively binds the A site of RI and the B site of type II.
The analog pair CPT-cAMP (selective for the B site of RII) and
N6-benzoyl-cAMP
(selective for the A site of either PKA isozyme) was used to
preferentially activate type II PKA (4). For the following experiments,
each analog was used at 50 µM, inasmuch as this concentration is
submaximal in eliciting changes in
Isc (data not
shown) yet capable of stimulating PKA activity.
Stimulation of chloride efflux by PKA type-selective analog pairs.
To compare the type of PKA activating CFTR in airway, intestinal, and
pancreatic cells, isotopic efflux assays were performed on these cells
in the presence of type-selective cAMP analog pairs. Capan-1, T84, and
Calu-3 cells were grown to confluency and equilibrated in
36Cl-containing HBR. After cells
were washed to remove extracellular chloride, the analog pairs were
added to chloride-free HBR (50 µM each analog), and aliquots were
replaced every 30 s to measure chloride efflux. For comparison,
responses to the analogs were quantitated by averaging the efflux rates
calculated for each 30-s time point over the 5 min in which the analogs
were present. In these experiments we chose concentrations of cAMP
analogs similar to those used in the literature (9, 26, 30). The type
II-selective pair, CPT-cAMP plus
N6-benzoyl-cAMP,
stimulated a sustained chloride efflux in all three cell types (Fig.
1, Table
1). The type I-selective analog pair, AHA-cAMP plus 8-PIP-cAMP, resulted in a quick pulse of chloride secretion in Calu-3 cells that is transiently larger than that seen
with the type II-selective analogs but leads to a smaller average rate
of efflux over the time period measured. The type I-selective analogs
did not stimulate significant efflux in T84 or Capan-1 cells.
Therefore, the type II-selective analog pair stimulated a larger
chloride efflux than the type I-selective pair in Capan-1 and T84
cells, but not in Calu-3 cells. Incubation of Calu-3 cells with the
calcium chelator BAPTA-AM before addition of the analogs blocked the
efflux stimulated by the type I-selective analog pair but had no effect
on that stimulated by the type II-selective pair (Fig.
2). Similarly, BAPTA-AM had no effect on
the effluxes stimulated by the type II-selective analogs in T84 or
Capan-1 cells. These results showed that the chloride efflux activated by the type I-selective analogs was calcium dependent in Calu-3 cells,
whereas that activated by the type II-selective analogs was not. The
efflux stimulated by the type II-selective analogs may even be
inhibited by calcium, inasmuch as there was a marked increase in this
efflux in the presence of BAPTA-AM in Capan-1 and T84 cells. The
results suggested that PKA II may activate a sustained,
calcium-independent chloride efflux in Capan-1, T84, and Calu-3 cells,
consistent with the activation of CFTR.

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Fig. 1.
Effect of protein kinase A (PKA) type-selective analog pairs on
36Cl
efflux from Capan-1 (A), Calu-3
(B), and T84
(C) cells. Cells were loaded with
36Cl
and washed, and aliquots of buffer were taken and replaced every 30 s
to measure Cl efflux.
Horizontal bars indicate presence of type I- or type II-selective
analog pairs at 50 µM each analog. Efflux was calculated as
percentage of Cl secreted
into buffer since previous time point and plotted as a function of
time. Error bars, SE (n 3).
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Table 1.
Effect of type-selective analogs on average rate of
36Cl efflux during stimulation in Capan-1,
T84, and Calu-3 cells
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Fig. 2.
Effect of calcium chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester (BAPTA-AM) on cAMP analog-stimulated
Cl efflux in Calu-3
(A and
B), Capan-1
(C), and T84
(D) cells. Cells were incubated with
10 µM BAPTA-AM for 30 min before assay. Horizontal bars indicate
presence of type-selective analogs at 50 µM each analog. Error bars,
SE (n 3). , Type I-selective
analogs; , type II-selective analogs; , type I-selective
analogs + BAPTA-AM; , type II-selective analogs + BAPTA-AM;
, control.
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Stimulation of changes in Isc by analog
pairs.
To compare efflux results with electrogenic chloride transport, we also
examined CFTR function by measuring the effect of the analogs on
Isc across
monolayers of the three cell types. Capan-1, T84, and Calu-3 cells were
grown to confluency on collagen-coated filters and mounted in modified
Ussing chambers. Chloride-free Ringer solution was placed on the apical
side of the filters to increase the driving force for transepithelial
chloride transport. Initial experiments were performed to determine the
concentration of type-selective analogs that had an effect on
Isc but did not maximally stimulate the current (data not shown), and as in the isotopic efflux assays, each analog was used at 50 µM. Addition of
type I-selective analogs, AHA-cAMP plus PIP-cAMP, to the basolateral side of the cells resulted in minor increases in
Isc (Table
2). When the type II-selective analogs
CPT-cAMP and
N6-benzoyl-cAMP
were added, 3- to 30-times larger currents were stimulated, depending
on the cell type. Addition of the
Na+-K+-2Cl
cotransporter blocker bumetanide (100 µM) to the basolateral side of
the cells decreased
Isc, consistent
with current due to chloride secretion. In each cell type the type
II-selective analogs stimulated a significantly larger increase in
Isc than did the type I-selective analogs (P < 0.05).
Interestingly, the type I-selective analogs did not activate a large,
transient increase in current analogous to the large, transient
increase in chloride efflux seen in Calu-3 cells, suggesting that the
increase in chloride efflux was not electrogenic or may be due to
differences in the culturing conditions of the cells for the two types
of experiments.
Inhibitor studies of the stimulation of
Isc in Capan-1 cells.
Our experiments showed that the type II-selective analogs stimulated a
sustained calcium-independent chloride efflux and activated larger
changes in Isc
than the type I-selective analogs in Capan-1, T84, and Calu-3 cells. To
determine whether the analogs were activating current consistent with
CFTR, Capan-1 cells were incubated with the chloride channel blockers
DIDS or glibenclamide before addition of the analogs. The amount of
current stimulated by the type I-selective analogs was small, and
incubation with 500 µM DIDS had an insignificant effect on current
(P = 0.16). Incubation with DIDS also
did not significantly affect current stimulated by the type
II-selective analogs (P = 0.22; Table
3). In contrast, incubation of Capan-1 cells with 500 µM glibenclamide before analog addition significantly decreased the current generated by type I- or type II-selective analogs. The inhibition of current by glibenclamide but not by DIDS was
consistent with the activation of current by the type II-selective
analog pair through CFTR.
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Table 3.
Effect of chloride channel blockers on Isc stimulated by
type I- or type II-selective analogs in Capan-1 cells
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Effect of nystatin permeabilization of the basolateral membrane in
T84 cells.
Although PKA activates CFTR chloride channels, an alternative
explanation of the above data was that the compounds used can affect
basolateral potassium channels, thereby influencing the driving force
for chloride secretion (1). To address this possibility, the
basolateral membranes were permeabilized, and the above experiments were repeated. T84 cells were mounted in modified Ussing chambers and
incubated for 10-30 min with 0.4-0.6 mg/ml nystatin applied to the basolateral side. Type II-selective analogs were then added, and
the change in Isc
was measured. There was no significant difference in current stimulated
by the type II-selective analogs between the nystatin-treated and
control T84 cells (Table 4). Bumetanide (100 µM) no longer decreased the current generated by the analogs in
the nystatin-treated cells, indicating that the basolateral membrane
had indeed been permeabilized. These experiments, in which ion channels
in the apical membrane were assayed directly, suggested that type
II-selective analogs stimulated current through an apical chloride
channel, presumably CFTR, in T84 cells.
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Table 4.
Effect of permeabilization of basolateral membrane of T84 cells on
Isc stimulated by type II-selective analogs
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Activation of PKA by type-selective analog pairs.
The differences seen with regard to stimulation of CFTR with type I-
vs. type II-selective analogs could be explained by the relative
effectiveness of the analogs to activate PKA, rather than a specific
ability to stimulate the different types of PKA. Capan-1 and T84 cells
were incubated with type I- or type II-selective analog pairs (50 µM
each analog, the same concentration of analogs used in the measurement
of Isc) for 15 min. Cells were washed and lysed, and the percentage of soluble PKA
activated by the analogs was measured using the PKA assay system (see
MATERIALS AND METHODS). Figure
3 shows the percentage of PKA activated by the analogs (i.e., amount activated / total activity in the soluble fraction). Type I- and type II-selective analog pairs activated soluble
PKA to a similar level in both cell types. These results suggested that
the difference in the ability of the type I- vs. type II-selective
analogs to stimulate CFTR was not due to the inability of the type
I-selective analog pair to stimulate soluble PKA.

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Fig. 3.
Stimulation of soluble PKA activity by type-selective analogs. Capan-1
and T84 cells were incubated with type I- or type II-selective analog
pairs (50 µM each analog), and percentage of PKA activated by analogs
in soluble fraction was measured using PKA assay system. Error bars, SE
(n = 3-4).
* P < 0.05 vs. control. I,
type I-selective analog pair; II, type II-selective analog pair.
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Effect of forskolin addition after type-selective analog pairs on
Isc.
An apparent paradox arose, in that the quantities of type I- and type
II-selective analogs inducing similar levels of PKA activity (Fig. 3)
stimulated significantly different amounts of Isc (Table 2).
Isc was again
measured with the addition of 10 µM forskolin after the type I- or
type II-selective analog pairs. Representative traces are shown in Fig.
4A. In all
three cell lines, addition of forskolin increased
Isc after type
I-selective analogs but had little additional effect on current when
added after the type II-selective analogs. The percentage of
Isc due to the
type-selective analogs compared with the total change in Isc (cAMP analogs
plus forskolin) was calculated (Fig.
4B). Type II-selective analogs
stimulated a significantly larger proportion of maximal current than
type I-selective analogs (P < 0.001)
in all three cell types. These results suggested that the type
II-selective analogs and forskolin were acting through the same pathway
to stimulate CFTR-mediated current, yet the paradox of how forskolin stimulated more PKA activity and increased current in cells pretreated with the type I-selective analog pair remained.

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Fig. 4.
Effect of addition of forskolin after type-selective analogs on
short-circuit current
(Isc) in
Capan-1, T84, and Calu-3 cells. A:
representative traces of
Isc in response
to type-selective analogs (50 µM each analog) followed by 10 µM
forskolin (F) and 100 µM bumetanide (B).
B: percentage of current due to each
analog pair compared with total current (analogs + forskolin). Error
bars, SE; number above bars is number of experiments.
* Significantly different from I
(P < 0.001).
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Activation of insoluble PKA by type-selective analog pairs.
The above data could be explained by differential activation of PKA in
cellular compartments, inasmuch as type II PKA can be targeted to
specific intracellular sites by proteins such as AKAPs. To test this
possibility, activation of insoluble PKA activity by the type I- vs.
type II-selective analogs was examined. Capan-1 cells were incubated
with type I- or type II-selective analogs, washed, lysed, and
fractionated into soluble and insoluble compartments. The percentage of
PKA activated by the analogs was then measured with the PKA assay
system, as described above. Although type I- and type II-selective
pairs activated ~100% of the soluble PKA (Fig. 3), the type
II-selective analogs stimulated approximately three times as much
insoluble PKA activity as did the type I-selective analogs (23.9 ± 16.3% in unstimulated cells, 22.1 ± 6.5% in type I-stimulated
cells, and 76.4 ± 14.0% in type II-stimulated cells, n = 3-4,
P < 0.05 for type I vs. type II;
Fig. 5). The amount of cAMP-activatable PKA
was ~10% less in the insoluble fraction than in the soluble
fraction, making the amount of insoluble PKA stimulated by the type
II-selective analogs small compared with the total amount of PKA that
can be activated in the whole cell (6.8% of the cellular PKA was
stimulated by the type II-selective analogs in the particulate
fraction, whereas the type I-selective analogs stimulated only 1.7%).
Therefore, the type I-selective analogs stimulated 100% of soluble PKA
activity but none of the particulate, whereas the type II-selective
analogs stimulated PKA activity in the soluble and insoluble fractions
of Capan-1 cells. These results suggested that type II PKA localized to
a specific insoluble compartment stimulated CFTR-mediated current.

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Fig. 5.
Stimulation of insoluble PKA activity in Capan-1 cells. Capan-1 cells
were incubated with type-selective analog pairs (50 µM each analog),
lysed, and fractionated into soluble and insoluble compartments.
Percentage of PKA activated by analogs in insoluble fraction was
measured with PKA assay system. Error bars, SE
(n = 3-4). * Significantly
different from control (P < 0.05);
** significantly different from I
(P < 0.05).
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DISCUSSION |
cAMP analogs selective for the A or B site of a specific regulatory
subunit of PKA can be paired to synergistically activate a specific
type of PKA, and a specific outcome of PKA activation may be measured.
This approach has been used to determine the role of PKA types in a
variety of systems. The use of type-selective analog pairs has
implicated type I PKA activity in the inhibition of human T lymphocyte
proliferation (26), in leutinizing hormone-stimulated steroidogenesis in rat Leydig cells (21), and in the control of
corticosterone production by zona fasciculata/reticularis cells (30),
whereas the activity of type II PKA has been shown to be predominantly
responsible for human placental renin secretion associated with
-adrenoceptor activation (9), growth inhibition of HL-60 human
promyelocytic leukemia cells (24), and aldosterone production by zona
glomerulosa cells (30). In our studies we applied type-selective pairs
of analogs to Capan-1, T84, and Calu-3 cells and measured the effect of
these analogs on CFTR-mediated chloride secretion. Although it was well
documented that PKA phosphorylates CFTR, the specific type of PKA
involved in various cell types had not been determined.
Application of the type II-selective analog pair CPT-cAMP plus
N6-benzoyl-cAMP
resulted in stimulation of chloride secretion as measured by isotopic
flux experiments or
Isc measurements
(Fig. 1, Table 2) in Capan-1, T84, and Calu-3 cells. The sustained nature of the efflux stimulated, the calcium independence of the efflux, the sensitivity of the current to specific chloride channel blockers, and the localization of the effect to a channel in the apical
membrane suggested that the chloride channel stimulated by the type
II-selective analogs was CFTR (Fig. 2, Tables 3 and 4). Although type
II-selective analogs stimulated a larger change in
Isc in all three
cell types, type I-selective analogs did have some effect on chloride
secretion in Capan-1 and Calu-3 cells. Current was activated in Capan-1
cells with the type I-selective analogs, and this current was at most
partially blocked by DIDS and fully blocked by glibenclamide. These
analogs may be stimulating CFTR by activating some type II PKA or
through a "spillover" effect of type I activating CFTR.
Alternatively, another apical channel may be stimulated. Similarly, the
type I-selective analogs activated a calcium-dependent chloride efflux
in Calu-3 cells that was not apparent with
Isc measurements.
In this case, the type I-selective analogs might be stimulating a
calcium-dependent chloride channel through the mobilization of calcium
by an increase in cAMP levels; the presence of this channel could be
culture dependent, inasmuch as Calu-3 cells are generally nonpolarized
in the efflux assays but are polarized on the permeable supports used
for the Ussing chamber experiments. Our experiments suggested that type
II PKA played the predominant role in CFTR-mediated chloride secretion in Capan-1, T84, and Calu-3 cells.
Type I- and type II-selective analog pairs were able to stimulate
soluble PKA activity to similar levels in Capan-1 and T84 cells (Fig.
3). This result seemed confusing in light of the fact that the analog
pairs had significantly different effects on chloride secretion. This
paradox was further complicated by the fact that addition of forskolin
after the type I-selective analogs significantly increased current,
whereas it had little additional effect after type II-selective analogs
(Fig. 4). This result suggested that the type II-selective analogs and
forskolin acted through the same pathway, inasmuch as they were not
additive, and that type I-selective analogs may overlap in effect but
were not identical to forskolin. Measurement of insoluble PKA activity
after incubation with the type-selective analog pairs provided an
explanation for this observation. At the concentrations used, type
II-selective analogs stimulated three times as much insoluble PKA
activity as did the type I-selective analog pair (Fig. 5). This result suggested that the stimulation of a specifically localized PKA activity
was important in CFTR-mediated chloride secretion.
Compartmentalization of PKA activity to enhance specificity and speed
of phosphorylation of substrates is not a novel idea (7, 20). Specific
proteins, AKAPs, have been isolated that bind to the RII subunit and
may localize type II PKA to specific regions of the cell, such as to
the cortical actin cytoskeleton by AKAP75 (18, 22) or to the
sarcoplasmic reticulum by AKAP100 (19). An AKAP has also been isolated
that not only binds type II PKA but also phosphatase 2B and, thereby,
localizes phosphorylating and dephosphorylating activities close to the
substrate to enhance response rates to stimuli (8). Although we have
not colocalized type II PKA with CFTR in this study, it is interesting
to speculate that the insoluble PKA activity stimulated by the type
II-selective analogs is situated close to CFTR, and only activation of
this compartmentalized PKA will phosphorylate CFTR.
 |
ACKNOWLEDGEMENTS |
This work was supported by grants from the Cystic Fibrosis
Foundation and by National Heart, Lung, and Blood Institute Grant HL-50160.
 |
FOOTNOTES |
Address for reprint requests: M. L. Drumm, Dept. of Pediatrics, Case
Western Reserve University, 10900 Euclid Ave., Cleveland, OH
44106-4948.
Received 21 April 1997; accepted in final form 4 December 1997.
 |
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