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(Received for publication, August 30,
1995; and in revised form, November 20, 1995) From the
Mechanisms of cross-talk between different classes of signaling
molecules are inadequately understood. We have used clonal Madin-Darby
canine kidney (MDCK-D
Extracellular ATP, an important extracellular signaling
molecule, is stored and released from sympathetic neurotransmitter
vesicles and from stressed/damaged cells. Receptors that interact
specifically with ATP (classified as P Interaction of extracellular nucleotides with P MDCK cells are a well differentiated epithelial cell line and have
been widely used as a model system for studying the regulation of
epithelial cell function(18, 19, 20) . In
these cells, cAMP regulates Cl
We assessed the ability of various P
Figure 1:
P
In general, P
Figure 2:
Activation of PKC does not mimic
nucleotide effect on cAMP production. MDCK-D
Figure 3:
Nucleotide- and PGE
Signaling pathways utilized by P To date, however, there is no
evidence for the interaction of P
Figure 4:
Nucleotides, arachidonic acid, and
PGE
Figure 5:
Inhibition of cyclooxygenase blocks
nucleotide- and arachidonic acid-stimulated cAMP production. a, cells were preincubated in the presence or absence of 1
µM indomethacin, a cyclooxygenase inhibitor, for 15 min
prior to incubation for 7 min with the indicated concentrations of
arachidonic acid. b, after a 15 min preincubation in the
presence or absence of 1 µM indomethacin, cells were
stimulated for 7 min with buffer, 100 µM ATP, 100
µM UTP, or 0.3 µM forskolin (Fsk).
The data represent the mean ± S.E. of triplicate determinations
from a representative experiment with similar results obtained in at
least three separate experiments.
Modulation of adenylyl cyclase activity
by extracellular nucleotides has been previously noted, but, in
general, the effect of nucleotides on cAMP accumulation is inhibitory.
Roles for G Our
results in MDCK-D Thus, extracellular nucleotides released
from granules or in other settings, such as cell damage, can regulate
cAMP formation in a variety of cell types. In many cases this effect is
inhibitory. However, depending on cell type, nucleotides can stimulate
cAMP formation by at least two different mechanisms. In aortic
endothelial cells, L cells, and adrenal medullary cells this
stimulation appears to be mediated by PKC. In MDCK-D
Volume 271,
Number 4,
Issue of January 26, 1996 pp. 2029-2032
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Purinergic Receptor Agonists Enhance cAMP Production in
Madin-Darby Canine Kidney Epithelial Cells via an Autocrine/Paracrine
Mechanism (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) epithelial cells as a model system
to investigate the effects of extracellular nucleotides (e.g. ATP, UTP), which promote increase in activity of several
phospholipases, on cAMP production. In contrast to observations in some
other cell systems, ATP and UTP, acting via P
purinergic
receptors, stimulated cAMP production in MDCK-D cells. At
maximally effective concentrations, ATP and UTP were not additive with
the 
-adrenergic receptor agonist isoproterenol, but were
synergistic with forskolin in increasing cAMP production, indicating
that G is activated by these nucleotides.
Additionally, we found that (a) nucleotide-induced increases
in cAMP were blocked by indomethacin, a cyclooxygenase inhibitor, (b) arachidonic acid increased cellular cAMP levels in an
indomethacin-sensitive fashion, and (c) PGE
, the
major metabolite of arachidonic acid, stimulated cAMP formation.
Overall, our results suggest a mechanism by which extracellular
nucleotides stimulate release of arachidonic acid which is metabolized
to PGE which, in turn, acts in an autocrine/paracrine
fashion via prostaglandin receptors to activate G and
increase cAMP. Based on the ability of extracellular nucleotides to
stimulate the formation and release of prostaglandins in MDCK-D
epithelial and other cells, we hypothesize that receptor-mediated
prostaglandin release may be a general mechanism that regulates cAMP
formation in many types of cells.
purinergic
receptors) are present in many tissues and cell
types(1, 2, 3, 4) . Two subclasses
of G protein-coupled P purinergic receptors include
P and P
receptors which differ in their
specificity for nucleotide. In particular, P
receptors
bind ATP, while P
receptors respond to both ATP and
UTP(2, 5) . Additionally, ATP can be metabolized to
adenosine, the agonist for P
purinergic receptors. purinergic receptors elicit a range of intracellular signaling
responses. The most common response elicited by ATP is the activation
of phospholipase C with subsequent increases in 1,4,5-inositol
trisphosphate formation, diacylglycerol production, increased
intracellular Ca, and activation of protein kinase C
(PKC) (
)(5, 6, 7, 8) .
Activation of phospholipase A and phospholipase D has also
been reported to occur in response to
nucleotides(9, 10, 11) . In addition, in rat
hepatocytes, FRTL-5 thyroid cells, mouse ventricular myocytes, and
C6-glioma cells, extracellular ATP inhibits cAMP production by both
pertussis toxin-sensitive and insensitive pathways (8, 12, 13, 14, 15, 16, 17) . ion secretion,
polarized vesicle transport, and Na
/K
ATPase
activity(21, 22, 23, 24) .
MDCK-D
cells, a clonal line derived from parental cells (25, 26) , appear to express both P and
P
purinergic receptors(10, 27) . Because
of the importance of cAMP in regulating epithelial cell function and
the potential interaction between P
purinergic receptor
response pathways and the adenylyl cyclase pathway, we examined the
effect of extracellular nucleotides on cAMP generation in MDCK-D cells. In contrast to observations in other cell lines, we found
that both ATP and UTP stimulated cAMP production in MDCK-D cells. Several mechanisms relating phospholipase activation to
modulation of cAMP accumulation could explain this result. In
particular, increases in Ca or PKC activity following
phospholipase C activation can alter adenylyl cyclase activity. In this
study, however, we demonstrate that nucleotide-induced phospholipase
activation leads to increased levels of cAMP by the release of
arachidonic acid, and in turn, cyclooxygenase-derived products. We
propose that utilization of this autocrine/paracrine pathway may be a
general mechanism for regulating cAMP production in epithelial cells
and other cell types.
Cell Culture
MDCK-D cells were
maintained in Dulbecco's modified Eagle's medium
supplemented with 10% serum as described
previously(25, 26) . Cells were used in assays at
60-80% confluence. At cell densities greater than this, basal
cAMP levels were increased.Measurement of cAMP Accumulation
Prior to
treatment of cells, growth medium was removed and cells were
equilibrated for 30 min at 37 °C in serum-free Dulbecco's
modified Eagle's medium containing 20 mM HEPES buffer
(DMEH; pH 7.4). Subsequently, cells were incubated in fresh
HEPES-buffered Dulbecco's modified Eagle's medium and
various agents as described in the figure legends. Unless otherwise
indicated, incubations with agonist were conducted for 5 min at 37
°C in the presence of 200 µM IBMX, a phosphodiesterase
inhibitor, and terminated by aspiration of medium and addition of 7.5%
trichloroacetic acid. Trichloroacetic acid extracts were frozen
(-20 °C) until assay. Intracellular cAMP levels were
determined by radioimmunoassay (Calbiochem) of trichloroacetic acid
extracts following acetylation according to manufacturer's
protocol. The sensitivity of this assay allowed for large dilution of
trichloroacetic acid extracts such that ether extraction of
trichloroacetic acid was unnecessary. Production of cAMP was normalized
to the amount of acid-insoluble protein assayed by the Bio-Rad protein
assay.Chemicals
Chemicals were purchased from the
following sources: forskolin and anti-cAMP antibody were from
Calbiochem. I-cAMP from DuPont NEN, and all other
reagents were from Sigma.
receptor and
P receptor agonists to affect cAMP accumulation in
MDCK-D cells. As shown in Fig. 1a, both ATP
and UTP stimulated cAMP formation. It is unlikely that this response to
ATP resulted from breakdown to adenosine and activation of P purinergic receptors, since adenosine failed to alter cAMP
production, whereas ATPS, a nonhydrolyzable ATP analog, stimulated
cAMP formation to the same extent as ATP. This suggested that
extracellular nucleotides acting at P
purinergic receptors
were mediating cAMP formation. The ability of UTP, a P receptor agonist, and 2-methylthio-ATP, a P
receptor
agonist, to increase cAMP indicate that both P
and
P
receptors are positively coupled to adenylyl cyclase
activation in MDCK-D
cells. P purinergic
receptor-mediated stimulation of cAMP formation was
concentration-dependent (Fig. 1b), exhibiting an
average EC for UTP and ATP of 4.2 ± 1.8 µM and 22.5 ± 1.1 µM, respectively. The ability
of nucleotides to stimulate cAMP production in MDCK-D
cells
occurred at concentrations previously shown to activate
phospholipase-linked pathways in this and other cell
systems(10, 12, 14, 28) . At
maximally effective concentrations, UTP-stimulated cAMP formation was
typically not as great as that occurring with ATP; a result consistent
with the interaction of ATP with multiple (presumably both P and P
) receptor populations.
receptor agonists increase cAMP in MDCK cells. MDCK-D cells were treated as described under ``Materials and
Methods.'' a, cells were incubated in the presence or
absence of the indicated nucleotide (100 µM), adenosine (Adn; 100 µM), or isoproterenol (Iso; 10
µM). b, cells were stimulated with the indicated
concentrations of ATP and UTP. Incubations were for 5 min in the
presence of 200 µM isobutylmethylxanthine. Cells were
treated and cAMP assayed as described under ``Materials and
Methods.'' Data represent the mean ± S.E. of triplicate
determinations from a representative experiment with similar results
obtained in at least three separate
experiments.
and P
purinergic receptors mediate the activation of
phospholipase C with subsequent increases cellular Ca
levels(5, 6, 7, 8) . Increased
intracellular Ca
is known to stimulate adenylyl
cyclase through a variety of mechanisms, including interaction with
calmodulin and activation of
PKC(29, 30, 31, 32) . Since ATP and
UTP increase intracellular Ca
ion concentration in
MDCK-D
cells(27) , it is possible that nucleotides
stimulate cAMP production by a Ca-dependent process.
Because Ca
/calmodulin-stimulated adenylyl cyclase
isoforms, types I and VIII, have not been detected in
kidney(33, 34) , it is unlikely that the
nucleotide-stimulated increase in cAMP in MDCK cells results from a
direct interaction of Ca
/calmodulin with adenylyl
cyclase. We therefore examined the extent to which activation of PKC
could mimic the effect of nucleotides on cAMP production in these
cells. As shown in Fig. 2, treatment of cells with the phorbol
ester, phorbol 12-myristate 13-acetate, for 10 min had no effect on
basal or forskolin-stimulated cAMP accumulation. Since protein kinase
C-dependent modulation of the activity of certain isoforms of adenylyl
cyclase is conditional upon G
activation(35, 36) , we examined the effect of
nucleotides on
-adrenergic receptor-stimulated cAMP production. As
shown in Fig. 3, ATP and UTP were not additive with the
-adrenergic receptor agonist, isoproterenol, in increasing cAMP
formation. It therefore appears that in MDCK-D cells, PKC
activation is not directly responsible for the nucleotide-mediated
increase in cAMP.
cells were
preincubated in the presence or absence of 50 ng/ml phorbol
12-myristate, 13-acetate (PMA), a PKC activator, for 10 min at
37 °C. Subsequently, isobutylmethylxanthine and either 100
µM nucleotide or 1 µM forskolin (Fsk) was added and incubations continued as described under
``Materials and Methods.'' The data represent the mean
± S.E. of triplicate determinations with similar results
obtained in multiple experiments.
-stimulated
cAMP production is synergistic with forskolin but not with
isoproterenol. Cells were stimulated with buffer, 100 µM nucleotide, 10 µM isoproterenol (Iso), 1
µM forskolin (Fsk), or combinations thereof. The inset shows cAMP formation in response to varying
concentrations of PGE in the presence or absence of 0.1
µM forskolin. The data represent the mean ± S.E. of
triplicate determinations from a representative experiment with similar
results obtained in at least three separate
experiments.
purinergic receptors on MDCK-D cells were
investigated further in experiments that examined cAMP formation
following the simultaneous addition of nucleotides with either
isoproterenol or forskolin. As shown in Fig. 3, when tested at
maximally effective concentrations, ATP and UTP were not additive with
each other or with the response elicited by isoproterenol. This lack of
additivity is consistent with the utilization of a common pathway by
both P purinergic receptors and -adrenergic receptors.
In contrast, nucleotide- and isoproterenol-stimulated cAMP production
were both synergistic with forskolin. As shown here in studies with
isoproterenol and PGE ( Fig. 3and inset)
and as demonstrated previously(37, 38, 39) ,
synergistic effects of such agonists and forskolin in stimulating cAMP
production is associated with activation of G,
probably by enhancing the ability of G
to interact
with adenylyl cyclase(39, 40) . Thus, it appears that
the ATP and UTP-mediated increase in cAMP production involves
G
activation.
purinergic receptors
directly with G
. We hypothesized therefore, that the
activation of G might be secondary to purinergic
receptor-mediated stimulation of arachidonic acid release and the
subsequent formation of arachidonic acid metabolites, perhaps those
derived from the action of cyclooxygenase. In this regard, we have
found that activation of phospholipases, in particular phospholipase
A
, in MDCK-D cells results in the release of
arachidonic acid and, in turn, to the formation of
PGE(41) . Furthermore, PGE release is
inhibited by indomethacin, a cyclooxygenase inhibitor(42) .
Since both ATP and UTP stimulate the release of arachidonic acid in
these cells(10, 27) , it seemed possible that P purinergic receptor-mediated cAMP production was secondary to the
metabolism of arachidonic acid. Indeed, as shown in Fig. 4,
arachidonic acid stimulated cAMP formation with a time course that
parallels that of nucleotide-mediated activation. For both exogenous
arachidonic acid and nucleotides, maximal cAMP levels were obtained by
5 min. The effect of arachidonic acid to increase cAMP formation
occurred at concentrations between 1 and 10 µM, and
importantly, the ability of arachidonic acid to stimulate cAMP
accumulation was completely inhibited by 1 µM indomethacin (Fig. 5a). This result led us to predict that
cyclooxygenase activity and presumably prostaglandin produced from
arachidonic acid might be responsible for the effect of ATP and UTP to
stimulate cAMP production. Results presented in Fig. 5b confirm this prediction in that nucleotide-stimulated cAMP
production, like that elicited by arachidonic acid, was inhibited by
indomethacin. Taken together, our results indicate that in MDCK-D cells cyclooxygenase products, most likely PGE,
produced following P purinergic receptor activation of
phospholipases mediate the cAMP response observed in response to
extracellular nucleotides.
show similar kinetics of cAMP production in
MDCK-D cells. Cells were stimulated with 100 µM nucleotide, 1 µM arachidonic acid (AA), or
0.1 µM PGE for the indicated period of time.
The dotted line represents cAMP production in unstimulated
cells. The data represent the mean ± S.E. of duplicate
determinations from two individual
experiments.
activation, Ca, and PKC have
been implicated in ATP-mediated inhibition of cAMP
production(8, 14, 16, 17) .
Notwithstanding these inhibitory effects on adenylyl cyclase, there are
reports of extracellular nucleotide-mediated increases in cAMP
formation. For example, in bovine aortic endothelial cells, ATP results
in a 3-fold increase in cAMP production and a greater than additive
increase when added together with forskolin(43) . The mechanism
by which ATP elicited this response appears to involve both PKC
activation and ATP interaction with methylxanthine-sensitive receptors.
Similarly, in L cells and adrenal medullary endothelial cells, ATP
sensitizes adenylyl cyclase to stimulation by hormones and
forskolin(28, 44) . Sensitization of adenylyl cyclase
to activation by G
-linked receptor agonists or
forskolin have been described for PKC-dependent modulation of distinct
adenylyl cyclase
isoforms(35, 36, 45, 46) . It
therefore seems likely that in aortic endothelial cells, L cells, and
adrenal medullary cells PKC mediates the ability of P
purinergic receptor agonists to increase adenylyl cyclase
activity. In Swiss 3T3 fibroblasts and human A431 epidermoid carcinoma
cells, however, ATP-stimulated cAMP production is sensitive to
cyclooxygenase inhibitors(47, 48) . This effect is
associated with the ability of ATP to stimulate mitogenesis in these
cells(48, 49) . In contrast to results presented here,
in both 3T3 and A431 cells there is an initial delay of 15 min before
cAMP increases are observed, and maximal cAMP accumulation is not
achieved until 45 min after addition of ATP. Therefore, it seems less
likely that nucleotide-mediated cAMP formation in 3T3 cells or A431
cells serves a role in rapid alterations of cellular responses. cells demonstrate that extracellular
nucleotides rapidly stimulate cAMP formation by a mechanism involving
the liberation of arachidonic acid, and production of prostaglandin,
presumably PGE. PGE then acts in an
autocrine/paracrine manner at a prostaglandin receptor to activate
G and subsequently adenylyl cyclase. The ability of
P
-nucleotide receptors to stimulate cAMP by this mechanism,
therefore, depends on several properties. First, phospholipases must be
activated and arachidonic acid must be released in quantities
sufficient for substantial prostaglandin formation. Second,
cyclooxygenase must be present and active in the time frame during
which arachidonic acid is available. Third, the cells must possess
prostaglandin receptors, which are positively coupled to adenylyl
cyclase. In MDCK-D cells, as well as in 3T3 and A431 cells,
these criteria have been fulfilled. We predict that other cell types
will show similar features. epithelial, fibroblast, and epidermoid carcinoma cells,
nucleotide-stimulated cAMP formation results from the
cyclooxygenase-dependent metabolism of arachidonic acid. In this way,
P receptors can regulate both rapid (e.g. secretion) and long term (e.g. mitogenesis)
cAMP-dependent cell functions.
),
clonal Madin-Darby canine kidney cells; ATPS, adenosine
5`-O-(thiotriphosphate); PGE
, prostaglandin
E.
-We thank Elizabeth Peters and David Dirig for
their initial contributions to this project, Rachel Mastandrea for help
in conducting cAMP assays, and Kelly Bell for technical assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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