1 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and 2 Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo, Norway
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ABSTRACT |
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Chloride exit
across the apical membranes of secretory epithelial cells is acutely
regulated by the cAMP-mediated second messenger cascade. To better
understand the regulation of transepithelial chloride secretion, we
have characterized the complement of cAMP-dependent protein kinase
(PKA) isoforms present in the human colonic epithelial cell line T84.
Our results show that both type I and type II PKA are present in T84
cells. Immunoprecipitation of
8-azido-[32P]cAMP-labeled
cell lysates revealed that the major regulatory subunits of PKA were
RI and RII
. In addition, immunogold electron microscopy showed that RII
labeling was found on membranes of the
trans Golgi network and on apical
plasma membrane. In contrast, RI
was randomly distributed throughout
the cytoplasm, with no discernible membrane association. Northern blot
analysis of T84 RNA revealed that C
was the predominantly expressed
catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to
preferentially activate either PKA type I or PKA type II in intact T84
cell monolayers. Maximal levels of chloride secretion (~100
µA/cm2) were observed for both
type I and type II PKA-selective analog pairs. Subsequent addition of
forskolin was unable to further increase chloride secretion. Thus
activation of either type I or type II PKA is able to maximally
stimulate chloride secretion in T84 colonic epithelial cells.
protein kinase A; kinase activation; kinase isoforms; adenosine 3',5'-cyclic monophosphate analogs
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INTRODUCTION |
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INCREASES IN INTRACELLULAR cAMP have been implicated in
the regulation of a variety of cellular functions, including cell proliferation, secretion of macromolecules, membrane turnover (5, 6),
and both anionic and cationic ion channel activity (16, 24, 35). With
the exception of direct activation of ion channels by cAMP, all known
cAMP-dependent events in eukaryotic cells are mediated through the
actions of the cAMP-dependent protein kinase (PKA). Mammalian PKA is a
tetramer composed of two catalytic (C) subunits, which phosphorylate
specific serine and threonine residues on target substrates, and two
regulatory (R) subunits, which bind cAMP and thus regulate catalytic
activity. Two major types of mammalian PKA, type I and type II (PKA I
and PKA II), were initially described by their pattern of elution from
DEAE-cellulose columns. These kinases were distinguished by the
presence of different R subunits, termed RI and RII. Through
biochemical studies and gene cloning, three isoforms of the C subunit,
C, C
, and C
, and four isoforms of the R subunits, RI
,
RI
, RII
, and RII
, have now been identified. In addition to
different biochemical and functional properties, several lines of
evidence support specific roles for the different PKA isoforms. RI
,
RII
, and C
represent the ubiquitous mRNA forms found in most
tissues, whereas mRNA for RII
seems to be cell and tissue specific
in its distribution, and its expression is hormonally regulated in
ovarian granulosa cells and testicular Sertoli cells (22, 29). RI
has so far been detected in several human tissues, with the highest
expression in the brain. The highest levels of C
mRNA have been
observed in the human prostate, intestine, brain, and testis, whereas
the message for C
has only been detected in human testis. In
addition, specific subcellular localization of PKA also supports the
notion that specific functions are assigned to each isoform. For
example, RI is found throughout the cytoplasm, whereas RII is localized to nuclei, nucleoli, the microtubule organizing center, the Golgi apparatus, and the plasma membrane (17, 27).
Activation of PKA occurs upon binding of four cAMP molecules, two per R subunit monomer, where the binding takes place on two dissimilar sites, designated site B and site A, in a positive cooperative manner. cAMP binding dissociates the holoenzyme, releasing active C subunit and dimers of the R subunit. Various cAMP analogs have been generated that display selectivity for either site A or site B. When two cAMP analogs, each selective for a different binding site, are added in combination to PKA in vitro, the enzyme is activated in a synergistic manner (2, 3, 15). Moreover, because the cAMP analog specificity of site A is different for the two PKA isozymes, the synergistic activation of the PKA I isozyme can be distinguished from the synergistic activation of the PKA II isozyme (2, 15).
Transcellular movement of chloride ions across epithelial cells is brought about by the concerted actions of several basolateral transporters accumulating chloride above its electrochemical equilibrium in the cytoplasm. Chloride then exits the cell through a cAMP-regulated chloride channel located in the apical membrane. The cAMP-regulatable chloride channel in secretory epithelia has been identified, cloned, and expressed. Mutations in the primary sequence of this channel, the cystic fibrosis transmembrane conductance regulator (CFTR), lead to the most common lethal genetic disease of Caucasians, cystic fibrosis. This disease is characterized by impaired transepithelial chloride transport in response to activation of the cAMP-mediated second messenger cascade (24, 31, 35). Primary sequence analysis of CFTR reveals at least 10 PKA consensus phosphorylation sequences, although several unidentified phosphorylatable sites are also present. Of the consensus phosphorylation sites, four sites (Ser-660, Ser-737, Ser-795, and Ser-813) appear to play a major role in the regulation of CFTR chloride channel activity. Indeed, there is now much compelling evidence to support the view that PKA regulation of chloride secretion has a direct effect on the CFTR chloride channel. Efforts in our laboratory are focused on understanding the molecular and protein-protein interactions surrounding the acute activation of transepithelial chloride secretion. The aim of our present studies was therefore to determine which PKA isoforms are present in the human colonic epithelial cell line T84 (a well-characterized model cell line of transepithelial chloride secretion). In addition, we have used site-selective cAMP analogs to synergistically activate PKA I and PKA II in intact T84 cells, with the aim of elucidating the role of each isozyme as a modulator of chloride secretion.
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MATERIALS AND METHODS |
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Chemicals. 8-Azido-[32P]cAMP was from ICN Pharmaceuticals (Costa Mesa, CA). cAMP analogs were obtained from BioLog (La Jolla, CA), and fresh solutions were made for each experiment by dissolving analogs in DMSO. Antibodies to PKA R subunits were generated against either purified R subunit or peptides derived from R subunit sequences and have been previously characterized (38, 39, 42). All other chemicals were from Sigma (St. Louis, MO) and were of reagent grade quality.
Cell culture. T84 cells were grown in DMEM-Ham's F-12 (1:1), 14 mM NaHCO3, and 10% fetal bovine serum. The cells were incubated in a humidified atmosphere containing 5% CO2 at 37°C. For measurements of short-circuit currents (Isc), T84 cells were seeded onto Costar Snapwell cell culture inserts (1.0 cm2), and the culture medium was changed every 48 h. Isc measurements were performed on filters after 14-21 days in culture (see Isc measurements).
Northern blot analysis.
RNA from T84 cells was extracted using guanidinium isothiocyanate and
purified by centrifugation through cesium chloride. RNA samples (20 µg) were fractionated on a 1.5% agarose gel containing 6.7%
formaldehyde (vol/vol) in 20 mM sodium phosphate buffer (pH 7.0) and
transferred to nylon membranes (Micron Separations, Westboro, MA). RNA was cross-linked to the filter using a
Stratalinker (Stratagene, La Jolla, CA) and probed with
32P-labeled human (h) C and
hC
cDNA probes. The PKA C subunit cDNA probes were obtained from a
human testis cDNA library. Radioactive labeling of cDNA probes (250 ng)
was performed by random primer extension using
[
-32P]dATP (3,000 Ci/mmol). Filters were prehybridized in 1× Denhardt's solution,
5× saline sodium citrate (SSC), 50 mM sodium phosphate buffer (pH
6.5), 0.1% SDS, 250 µg/ml salmon sperm DNA, and 50% (vol/vol)
formamide at 42°C for 2 h, followed by hybridization at 42°C
overnight (106 cpm cDNA probe/ml
buffer). Filters were washed four times with 2× SSC-0.1% SDS at
room temperature (20 min each), followed by two washes at 65°C (20 min each) with 0.1× SSC-0.1% SDS. After washes, the filter was
subjected to autoradiography. Messenger RNA sizes were estimated by
comparison to RNA standards.
Photoaffinity labeling of R subunits with 8-azido-[32P]cAMP. Covalent incorporation of the cAMP analog 8-azido-[32P]cAMP was performed by incubating 200 µg protein from a cell lysate in a reaction mixture (final volume 90 µl) containing 50 mM MES (pH 6.2), 10 mM MgCl2, and 1 µM 8-azido-[32P]cAMP, in the presence or absence of 200 µM cAMP. Reactions were carried out for 60 min in the dark at 4°C in the presence or absence of unlabeled cAMP (100 µM). Covalent incorporation was accomplished by exposure of the reactions to ultraviolet light (254 nm), and the samples were irradiated for 10 min at 20°C.
Immunoprecipitations. Samples were adjusted to a volume of 200 µl in buffer containing PBS, 2 mM EGTA, 2 mM EDTA, 2.5% Triton X-100, 1 mM benzamidine, 2 µg/ml leupeptin and pepstatin A, and 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Staphylococcus aureus cells expressing protein A (Pansorbin cells, Calbiochem, San Diego, CA) were added to the samples, which were incubated by shaking for 20 min at 20°C and thereafter centrifuged (12,000 g for 3 min). Supernatants were transferred to new tubes, and antibody was added. Samples were shaken for 90 min at 4°C, after which S. aureus cells were added, and the incubations continued for a further 60 min. Antigen-antibody complexes bound to protein A were pelleted (12,000 g for 30 s), and pellets were washed twice. Pellets were resuspended in Laemmli sample buffer and resolved by SDS-PAGE using 3.5% stacking gels and 7.5% separating gels.
Cell fractionation. Confluent monolayers of cells were homogenized in a Dounce type homogenizer [10 mM Tris · HCl (pH 7.4) containing 1 mM EDTA, 250 mM sucrose, 1 mM benzamidine, 2 µg/ml leupeptin and pepstatin A, and 0.5 mM AEBSF]. Postnuclear supernatants (5,000 g for 10 min) were centrifuged at 110,000 g for 60 min to yield cytosolic and total membrane fractions. Cytosolic fractions were mixed with Laemmli SDS sample buffer, and the membrane pellets were resuspended in homogenization buffer and recentrifuged (110,000 g for 60 min) before solubilization in sample buffer.
Immunoblot analysis. Samples were resolved by SDS-PAGE and transferred to nitrocellulose. After block of remaining binding sites (Blotto), membranes were incubated overnight at 4°C with the appropriate antibodies. After extensive washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies, and the signal was visualized by enhanced chemiluminescence. Samples were exposed to X-ray film for equal amounts of time.
Electron microscopy.
After fixation in 2% paraformaldehyde-0.01% glutaraldehyde, the cells
were scraped from the dish and embedded in 2% gelatin before sucrose
infusion. Subsequently, the samples were diced into 1- to 2-mm cubes
and mounted on cutting stubs, shock frozen, and stored in liquid
nitrogen. Thin sections (70-100 nm) were cut using a Reichert
Ultracut S ultramicrotome with an FC4S cryoattachment. Sections were
lifted in a small drop of sucrose, mounted on Formvar-coated carbon
grids, and washed three times in PBS containing 0.5% BSA and 0.15%
glycine (buffer
A; pH 7.4). This was followed by a
30-min incubation with purified goat IgG (50 mg/ml) at 25°C and
three additional washes with buffer
A. All of the preceding steps were designed to ensure minimal nonspecific reaction to the antibodies used.
Sections were then incubated for 60 min with primary antibody (either a
rabbit polyclonal antibody directed to RII or a murine monoclonal
antibody directed to RI
), followed by three washes in
buffer
A and a 60-min incubation in
gold-labeled second antibody (1-2 mg/ml). The sections were then
washed six times (5 min/wash) in
buffer
A and washed thoroughly in
buffer
A (5 changes) and in PBS (3 changes),
followed by a brief fixation step in 2.5% glutaraldehyde in PBS to
ensure that antibodies did not dissociate. Subsequent steps were three
further washes in PBS, five washes in water, and counterstaining with
uranyl acetate and embedment in 1.25% methylcellulose. Observation was
with a Jeol 1210 electron microscope.
Isc measurements.
T84 cells were grown in Snapwell filters (Costar), and used 14-20
days after seeding. All filters were fed the day before being used.
Filters were mounted in the Costar vertical chamber and voltage clamped
(Iowa Bioengineering; model 558C-5) using a 5-mV bipolar pulse to
estimate transepithelial resistance. Monolayers were bathed in a
bicarbonate-buffered saline (in mM: 145 NaCl, 5 KCl, 4.2 K2HPO4,
1.2 MgCl2, and 1.2 CaCl2) and continuously gassed
with 95% O2-5%
CO2. Monolayers were equilibrated
for at least 15 min before addition of cAMP analogs. Incremental
additions of cAMP analogs were made only after a
steady-state level of chloride secretion was attained with the previous
analog concentration. For paired analog additions, monolayers were
equilibrated for 15-20 min in the presence of the site B-directed
cAMP analog
[N6-monobutyryladenosine-3',5'-cyclic
monophosphate
(N6-MB-cAMP),
N6-benzoyladenosine-3',5'-cyclic
monophosphate
(N6-Bzl-cAMP), or
5,6-dichlorobenzimidazole riboside-3',5'-cyclic monophosphorothionate, Sp isomer
(Sp-5,6-DCl-cBIMPS)]; control monolayers received vehicle (DMSO) for the same incubation period. Site
A-directed cAMP analogs were added 20 min later at the indicated concentrations.
Isc were measured
when values had reached a new plateau (usually 10-15 min after the
addition of cAMP analogs). Analogs were added to both the mucosal and
serosal solutions. The T84 monolayers had resistances of 500-2,000
/cm2. Data were digitized with
the aid of a Gould Smartcase recorder at a sample frequency of 10 Hz
and saved to a computer hard drive. Changes in
Isc were
calculated as a difference current between the sustained phases of the
response and their respective baseline values.
Protein concentrations. Protein concentrations were determined by the method of Bradford using BSA fraction V as standard.
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RESULTS |
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Identification of PKA subunits in human colonic epithelial cells.
To resolve which of the C subunits of PKA are expressed in human
colonic epithelial cells, mRNA extracted from T84 cells was subject to
Northern blot analysis. The cloned cDNAs for hC hC
, and hC
were used to identify mRNAs for the various C subunits in T84 colonic
epithelial cells. The hC
-specific probe clearly identified a single 2.8-kb mRNA from T84 cells that comigrates with
previously studied C
transcripts (Fig. 1). Using the
hC
probe, no 1.8-kb C
mRNA was detected, but, in agreement with earlier studies showing that C
, C
, and C
cDNAs are capable of
cross-hybridizing with each of the C subunit mRNAs, the C
cDNA
hybridized to a single 2.8-kb mRNA, representing human C
. Interestingly, in addition to the 2.8-kb C
transcript, a 4.4-kb mRNA
corresponding to the size of the C
transcript was detected by the
hC
probe. However, detection of the hC
transcript using a C
or
C
probe required long exposure times to detect (~2 wk; data not
shown). Thus, although a small amount of C
may be present in T84
cells, it represents a very minor component of the PKA C subunit pool.
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Intracellular distribution.
Previous studies have documented an unequal distribution and expression
of PKA I and PKA II in many cell types. We thus monitored the
intracellular distribution of PKA in T84 cells. First, we performed
immunoblot analyses of both cytosolic and membrane fractions using
isotype-specific regulatory domain antibodies. Immunoblot analysis
confirmed what was observed for
8-azido-[32P]cAMP
labeling, that RI and RII
were the dominant PKA isoforms. Densitometric analysis of the immunoblots revealed that ~75% of RI
was in the soluble fraction, with ~25% associated with the membrane fraction (Fig. 3). Type RI
PKA
was found only in the cytosol, with no signal detectable in the
membrane fraction (Fig. 3). In contrast, >90% of RII
was found
associated with the membrane fraction, and <10% was associated with
the soluble fraction (Fig. 3). RII
was barely detectable in the
membrane fraction and was undetectable in the soluble fraction (Fig.
3). We further investigated the subcellular distribution
of PKA isoforms by immunogold electron microscopy, focusing on the
dominantly expressed PKA isoforms, namely
types I and II. Results
(Fig.
4D) show
that RI
was distributed randomly throughout the cytosol and showed
no specific association for the intracellular membrane systems. By
contrast, cells labeled using RII
antibodies showed label associated
with intracellular membranes, including label in the most
trans elements of the Golgi (Fig. 4,
A and
B) and the
trans Golgi network (TGN; Fig.
4B), in intracellular spherical
vesicles (Fig. 4C), and at the
plasma membrane (Fig. 4E).
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Effect of cAMP analogs on chloride secretion. Previous studies have demonstrated that binding of a cAMP analog selective for either site A or B on the R subunits of PKA stimulates binding of a cAMP analog selective for the other site, and two such site-selective analogs in combination demonstrate synergism in kinase activation. Thus the use of site-selective analog pairs can, to a certain extent, distinguish between effects mediated by PKA I and II. We selected cAMP analog pairs that complement each other in the synergistic activation of either PKA I or PKA II. Figure 5A shows a representative experiment for the activation of PKA II using the site-selective analogs Sp-5,6-DCl-cBIMPS (a PKA II site B-selective analog) and N6-phenyladenosine-3',5'-cyclic monophosphate (N6-Phe-cAMP; a PKA II site A-selective analog). Cells were pretreated with either DMSO (as vehicle control) or Sp-5,6-DCl-cBIMPS at a concentration (400 nM) that results in only marginal chloride secretion (~8 µA/cm2), before exposure to N6-Phe-cAMP at increasing concentrations. Forskolin (2 µM) was added at the end of the experiment to determine the maximum level of chloride secretion (~112 µA/cm2). Cells pretreated with Sp-5,6-DCl-cBIMPS showed a leftward shift in the N6-Phe-cAMP dose-response curve (EC50 for N6-Phe-cAMP alone, 31 µM; EC50 for N6-Phe-cAMP in the presence of 400 nM Sp-5,6-DCl-cBIMPS, 8.5 µM) consistent with a synergistic activation of PKA II by the two analogs in combination (Fig. 5B). Similar experiments were performed for PKA I activation using N6-MB-cAMP or N6-Bzl-cAMP as type I and type II site A priming analogs with increasing concentrations of 8-(6-aminohexylamino)adenosine-3',5'-cyclic monophosphate (a PKA I site B-selective analog). Table 1 shows the synergism quotients for a series of PKA I and PKA II isoform-selective cAMP analog pairs. For all analog pairs, the synergism quotients were >1 at low analog concentrations. The extent of synergism was optimal when the sum of the single analog responses was ~30% of the maximum. As the total analog concentration was increased beyond the optimum, the synergism quotient decreased. Because the cAMP analogs used are site selective, and not site specific, high concentrations of a single analog are able to stimulate secretion on their own. Once a maximal level of chloride secretion has been obtained, adding other cAMP analogs or even raising cAMP with forskolin is unable to elicit any increase in chloride secretion.
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DISCUSSION |
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There are two major isozymes of PKA, designated type I and type II. In addition to different biochemical properties, several lines of evidence support specific roles for each of the PKA isozymes in cellular responsiveness. For example, activation of PKA I, but not PKA II, in human T lymphocytes inhibits cell replication. Moreover, it is becoming increasingly clear that phosphorylation events are controlled not only by the relative activities of kinases and phosphatases but also by the subcellular localization of these enzymes within the cell. For example, the subcellular targeting of a multifunctional serine/threonine kinase, such as PKA, would enhance the selectivity of PKA by favoring its accessibility to specific substrate proteins. Indeed, there is evidence for subcellular targeting of both PKA I and PKA II. In the present study, we have investigated the levels and subcellular distribution of PKA subunits and the extent to which PKA I or PKA II may mediate the stimulatory effects of increased cAMP on chloride secretion from human epithelial cells.
We initially characterized the isoforms of PKA present in T84 cells by
investigating the presence of the subtypes of both the C and R
subunits. Human colonic epithelial cells express predominantly the mRNA
for the C subunit C, although a small amount of C
could be
detected on longer exposure of the Northern blots. The C
subunit has
been shown previously to represent the C subunit found in most tissues.
In contrast, the C
subunit has so far been located mainly in the
brain. At present we do not know whether the small amount of C
identified in T84 cells represents a physiologically important pool of
C subunit. Our results from
8-azido-[32P]cAMP
labeling and immunoprecipitations revealed that RII (specifically RII
) was the major R subunit in human colonic epithelial cells (~58%), whereas RI (RI
) constituted most of the remaining R
subunit. Although present in much smaller amounts, RI
and RII
were nonetheless detectable, contributing 8% and 2% of the total R
subunit population, respectively. We do not know whether the small
amount of RI
and RII
present in T84 cells represents a
physiologically important pool of R subunits. Two classes of cAMP
binding proteins have been reported in bovine and rat liver, one class
derived from PKA and a second class characterized by its ability to
bind a whole range of adenine analogs (especially adenosine). The
absence of an effect of 100 µM adenosine on the photoaffinity
labeling from colonic epithelial cells suggests that the adenosine
analog binding protein was not a target for the photoaffinity label
8-azido-[32P]cAMP.
Similarly, a lack of effect by the phosphodiesterase inhibitor IBMX on
8-azido-[32P]cAMP
incorporation indicates that enzymatic hydrolysis of
8-azido-[32P]cAMP or
cAMP did not affect the experimental results.
Immunoelectron microscopy of T84 cells using antibodies directed
against either RI or RII
revealed a striking difference in
subcellular localization between the two R subunits. Thus RI
is
randomly distributed throughout the cytosol with no specific membrane
association, whereas RII
is very intimately associated with
membranes of the TGN and the apical plasma membrane. In addition, RII
labeling is also seen on spherical vesicles enclosing an electron transparent space, consistent with early endosomes. These findings are in agreement with previous studies in other polarized epithelia and nonpolarized cells, which show localization of RII to the
apical plasma membrane, clathrin-coated pits, early endosomes, and the
TGN. It is thus clear that RII is localized to those compartments that
are active in endocytosis from the apical plasma membrane and in
recycling of cell surface proteins. It is interesting to speculate that
the subcellular localization of RII, and hence PKA II, may reflect a
specific function of the kinase in regulating the subcellular
distribution and activity of certain plasma membrane proteins. Support
for this view comes from increasing evidence that
phosphorylation-dephosphorylation plays a critical role in regulating
the activities and surface expression of a wide variety of receptors.
Indeed, such subcellular localization of PKA to clathrin-coated pits
and endocytic vesicles may in part explain our observations on the
PKA-dependent inhibition of endocytic activity in polarized epithelial
cells and the cAMP-dependent trafficking of CFTR chloride channels (30,
41). Accumulating evidence suggests that localization of PKA to
specific subcellular compartments can be brought about through the
interaction of PKA with specific anchoring proteins (AKAPs) (34, 36,
37). These anchoring proteins serve to tether PKA adjacent to specific
substrates, enhancing the specificity of the phosphorylating activity
of activated PKA. It will be of interest to determine whether such
anchoring proteins are present in T84 cells and, if so, whether they
are present in the same subcellular domain as CFTR.
To investigate whether one or both of PKA I and PKA II participate in mediating agonist-induced chloride secretion, we employed site-selective cAMP analogs on T84 human colonocytes to stimulate transepithelial chloride secretion. An accurate reflection of the relative roles of PKA I and PKA II in stimulating chloride secretion can be obtained with the use of site-selective analog pairs. The use of site-selective cAMP analog pairs to determine kinase isozyme activation relies on the principle of synergism between the cAMP binding sites of the R subunit. Although the R subunits of PKA I and II are homologous proteins (exhibiting the strongest homology in the cAMP binding sites), PKA I and II exhibit kinetic differences in cAMP analog specificities for binding. This difference in binding affinity, together with the positive cooperative binding interactions between the intrasubunit binding sites, can be exploited to differentiate between the isozymes in vitro and in intact cells. For example, site-selective analog pairs have proven useful in the elucidation of PKA isozyme regulation of processes including inhibition of T-lymphocyte proliferation and adipocyte phosphorylase activation. Our results (Table 1) show synergism in activation of chloride secretion from both type I and type II cAMP analog pairs, suggesting that activation of either PKA I or PKA II can lead to increased chloride secretion. It should be noted, however, that the selectivities of the various cAMP analogs for sites A and B on each kinase isozyme are normally expressed relative to the binding of cAMP at those sites. Thus the binding of various cAMP analogs is selective but not necessarily specific. At high concentrations, all analogs will bind to both sites, but they bind selectively to one or the other site at relatively low concentrations. For this reason, optimal synergism is demonstrated at low levels of cAMP analog stimulation. Indeed, optimum synergism between isozyme-selective analog pairs was observed when the sum of responses for the single analogs was between 25 and 30% of the maximum. These results are consistent with observations of PKA-mediated cell responses in several other tissues stimulated with pairs of site-selective cAMP analogs. For example, PKA I-directed analog pairs show maximum synergism in activating phosphorylase in both neutrophils and hepatocytes when the sum of the responses for the single analogs is ~20% of the maximum. Similar observations were also obtained for PKA II-mediated lipolysis in intact adipocytes. Because the mechanism of synergism of intact cell responses using site-selective analog pairs occurs via the exclusive activation of PKA, our observations are consistent with the hypothesis that activation of either PKA I or PKA II can stimulate increased transepithelial chloride secretion.
It is unlikely that any synergism is due to analog-stimulated elevation of endogenous cAMP. Rather, cAMP analogs have been shown to lower endogenous cAMP levels through an activation of phosphodiesterase(s) via protein-kinase-mediated short-term feedback. Another potential concern is the use of 8-(4-chlororophenylthio)adenosine-3',5'-cyclic monophosphate, which in addition to activating PKA II can also result in the activation of type II cGMP-dependent protein kinase (PKG II). Indeed, there have been reports that application of PKG II to excised membrane patches from natively expressing and CFTR-transfected cells can cause the activation of CFTR-mediated chloride transport (18, 26). It is also worth noting that the heat-stable enterotoxin from Escherichia coli (Sta) has been shown to activate guanylyl cyclase in T84 cells, with subsequent increase in chloride secretion. However, subsequent studies have shown that T84 cells do not express PKG II and that cGMP-mediated increases in chloride secretion are due to cross-activation of PKA.
We have demonstrated the presence of both PKA I and PKA II isozymes in
human colonic epithelial cells; PKA I accounted for approximately
one-third of the total PKA activity, and PKA II accounted for the
remaining two-thirds. Although both PKA isozymes are present, they show
clear spatial separation within the cell. Thus PKA I is soluble and
randomly distributed throughout the cytosol. In contrast, PKA II is
predominantly membrane associated. Use of synergistic pairs of
site-selective cAMP analogs demonstrated that activation of either
kinase isozyme was capable of activating a chloride secretory current.
However, the precise physiological role of these isozymes in this
process remains to be elucidated. For example, it is not known whether
different PKA isozymes preferentially activate different membrane
microdomains. Thus although activation of apical membrane CFTR chloride
channels (via phosphorylation) can lead to chloride secretion, it is
also known that activation of basolateral potassium channels alone can
produce chloride secretion by membrane hyperpolarization that increases
the driving force for apical chloride exit. It is therefore possible
that stimulation of chloride secretion by isozyme-selective cAMP analog
pairs may result from activation of different proteins or different
pools of the same protein. Thus future experiments will need to
be designed to elucidate the interactions between PKA I
and PKA II and activation of apical and/or basolateral
components of the secretory machinery. Finally, the results presented
make use of exogenous permeable cAMP analogs, a technique which
is likely to cause a uniform distribution of these
analogs throughout the cell. Because it has been shown that hormonal
agonists can lead to increases in cAMP in specific subcellular
compartments (1, 21), it is likely that the normal regulators of
secretion (e.g., -agonists, vasoactive intestinal peptide) could
lead to a localized accumulation of cAMP, resulting in specific isotype
activation.
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ACKNOWLEDGEMENTS |
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We thank Maitrayee Sahu and Raymond Carter for excellent technical assistance. We also thank Dr. J. D. Corbin for helpful discussion.
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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-47850.
Address for reprint requests: N. A. Bradbury, Dept. of Cell Biology and Physiology, S311 Biomedical Science Tower South, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
Received 6 August 1997; accepted in final form 4 May 1998.
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