* CIHR Group on the Molecular Mechanisms of Inflammation, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, CHUQ et Université Laval, Départements
d’Anatomie-Physiologie et
Médecine, Québec, Canada
Correspondence: Sylvain G. Bourgoin, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, CHUQ, 2705 Blvd. Laurier, Sainte-Foy, Québec, G1V 4G2, Canada. E-mail: Sylvain.bourgoin{at}crchul.ulaval.ca
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: protein kinase C • phosphodiesterase • ADA • cAMP
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenosine acts by interacting with specific receptors on the cell surface [19 ]. Studies investigating the structure-activity relationship of adenosine derivatives at A1, A2a, A2b, and A3 receptors have defined the A2a receptor subtype as the adenosine receptor mediating the suppression of neutrophil functional responses [1 ]. Although A2a receptor occupancy does not affect chemoattractant-stimulated generation of inositol 1,4,5-triphosphate by phospholipase C (PLC) [20 ] or the mobilization of intracellular pools of calcium [21 ], it decreases fMLP-mediated actin polymerization [5 ]. Furthermore, we recently demonstrated that the suppression of fMLP-mediated phospholipase D (PLD) activation by adenosine was correlated with the inhibition of the translocation of protein kinase C (PKC) and of Arf1 and RhoA small GTPases to neutrophil membranes [15 ]. Although the nature of the intracellular messenger mediating the inhibition of PLD activity and small GTPases activation by adenosine remains unclear, cyclic adenosine monophosphate (cAMP) analogues and agents that stimulate cAMP accumulation have been shown to inhibit fMLP-mediated stimulation of PLD [22 , 23 ].
Several signaling pathways, including PLD activation, initiated by receptor-ligand interactions are involved in the regulation of the oxidative burst [24 ]. A2a receptors are coupled to activation of adenylyl cyclase through the GTP-binding protein Gs [25 ], and their occupancy by adenosine has been shown previously to raise cAMP levels in human neutrophils [21 , 26 ]. Inhibition of superoxide generation by adenosine [27 ] and its enhancement by inhibitors of phosphodiesterase (PDE) support the hypothesis that cAMP may contribute to suppress this neutrophil function [1 , 28 ]. However, inhibitors of cAMP-dependent protein kinase A (PKA) have been shown to reverse the inhibitory effect of cAMP analogues but not of A2a or ß-adrenergic receptor agonists on the oxidative burst [25 ]. The cAMP-independent activation of a serine/threonine phosphatase by adenosine has been suggested to uncouple fMLP receptors from the signal transduction mechanisms [29 ].
In the present study, we have evaluated the effect of adenosine A2a
receptor occupancy on cAMP levels and of various pharmacological
modulators of the cAMP/PKA signaling pathway on the ability of
adenosine to suppress fMLP-mediated PLD activation. Together, the data
indicate that extracellular adenosine markedly potentiated fMLP-induced
cAMP elevation in neutrophils. Adenosine had no impact on the initial
elevation of cAMP levels induced by fMLP but produced a more persistent
stimulation of adenylyl cyclase. A2a receptor occupancy increased the
cAMP response induced by fMLP with a concomitant inhibition of PLD
activity and recruitment of RhoA and Arf to membranes. Our results
suggest that adenosine regulates fMLP-induced PLD activation via a
cAMP/PKA-dependent inhibition of the recruitment of small GTPases and
of PKC
to neutrophil membranes.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and RhoA antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal
Arf1 antibody was described in previous studies [30
].
Adenosine 3',5'-cyclic monophosphorothioate acetoxymethylester Sp
(Sp-cAMPs-AM) and Rp (Rp-cAMPs-AM) isomers were purchased from Biolog,
Life Science Institute (La Jolla, CA).
1-O-[3H]alkyl-2-lyso-phosphatidylcholine was
obtained from Amersham Pharmacia Biotech (Baie d’Urfé,
Québec, Canada).
Neutrophil purification
Venous blood was collected from healthy adult volunteers in
isocitrate anticoagulant solution. Neutrophils were separated as
described previously [15
]. Briefly, whole blood was
centrifuged at 180 g for 10 min, and the resulting platelet-rich
plasma was discarded. Leukocytes were obtained following erythrocytes
sedimentation at 1 g in 2% Dextran T-500. Mononuclear cells were
removed by centrifugation on Ficoll-Paque cushions, and contaminating
erythrocytes in the neutrophil pellets were removed by a 20-s hypotonic
lysis in water. Neutrophils were resuspended in Hanks’ balanced salt
solution (HBSS), pH 7.4, containing 0.8 mM Ca2+
but no Mg2+.
cAMP measurements
Neutrophils (107 cells/ml) were preincubated with 10
µM Ro20-1724 for 20 min at 37°C. Where indicated, 0.1 U/ml ADA was
also added to the cell suspensions to prevent the rapid accumulation of
adenosine in the extracellular milieu with time as described previously
[8
, 15
]. Then, cell suspensions
(1.5x107 cells) were preincubated 5 min with 10 µM CB
and the indicated concentrations of adenosine analogues or an equal
volume of the vehicle. Cells were stimulated with 0.1 µM fMLP for the
indicated times. Incubations were stopped by centrifuging the cell
suspensions at 5000 g for 1 min and mixing the cell pellets with
0.5 ml ice-cold acidified ethanol (99% ethanol, 20 mM HCl) and kept at
4°C. Samples were sonicated for 20 s and centrifuged at 13,000 g
for 45 min at 4°C. The supernatants were collected, dried under
nitrogen, and stored at -20°C until used for the cAMP measurements.
cAMP measurements were performed by Solution Recherche (Sainte-Foy,
Québec, Canada) using a radioimmunoassay purchased from
Diagnostic Products Corporation (Los Angeles, CA).
PLD measurements
Neutrophils were labeled with
1-O-[3H]alkyl-2-lyso-phosphatidylcholine (2
µCi/107 cells) for 90 min as described previously
[15
]. Neutrophils were washed and resuspended at
107 cells/ml. Cell suspensions (0.5 ml) were preincubated
at 37°C for 5 min and pretreated for 5 min with 10 µM CB in the
presence or absence of 0.1 U/ml ADA and the indicated concentrations of
adenosine analogues. Neutrophils were stimulated with 0.1 µM fMLP for
10 min in the presence of 1% ethanol. Incubations were stopped by
adding 1.8 ml chloroform/methanol/HCl (50:100:1, vol/vol/vol) and
unlabeled phosphatidylethanol (PEt) as a standard. Lipids were
extracted and dried under nitrogen. The lipid extracts were dissolved
in 40 µl chloroform/methanol (2:1, vol/vol) and spotted on prewashed
silica gel 60 thin-layer chromatography plates. [3H]PEt
was separated from the other lipids using the solvent mixture
chloroform/methanol/acetic acid (65:15:2, vol/vol/vol). Lipids were
visualized by Coomassie brilliant blue staining (0.03% dye, 35%
methanol, and 200 mM NaCl), and the different lipid classes were
scraped off the plates. Radioactivity in PEt was monitored by liquid
scintillation counting, and the results were corrected for background
radioactivity and quenching.
Translocation assays
Neutrophils (5x107 cells/ml) were treated with 1.1
mM DFP for 30 min at 24°C. Cell suspensions were centrifuged and
resuspended in HBSS at 107 cells/ml. The cells were
preincubated 5 min at 37°C and treated with 10 µM CB. Where
indicated, 0.1 U/ml ADA and/or adenosine analogues were added to the
cell suspensions 5 min prior to stimulation. Neutrophils were
stimulated with 0.1 µM fMLP for 2 min at 37°C. Incubations were
stopped by diluting the cells 1:5 with ice-cold HBSS, and the samples
were processed as described previously [15
]. Briefly,
cell suspensions were centrifuged as indicated and resuspended at
1.6 x 107 cells/ml in ice-cold KCl-Hepes relaxation
buffer [50 mM Hepes, 100 mM KCl, 5 mM NaCl, 1 mM MgCl2,
0.5 mM
ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic
acid (EGTA), 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 2.5 mM
phenylmethylsulfonyl fluoride (PMSF), adjusted to pH 7.2]. Cell
suspensions were sonicated for 20 s and centrifuged for 7 min at
700 g. Unbroken cells and nuclei were discarded, and the
supernatants were ultracentrifuged at 180,000 g for 45 min in a Beckman
TL-100 ultracentrifuge using a TL-100.4 rotor. Membrane pellets were
washed once and resuspended in a small volume of buffer A containing
0.25 M Na2HPO4, 0.3 M NaCl, 2.5% sodium
dodecyl sulfate (SDS), 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin,
and 2.5 mM PMSF, and samples were assayed for protein content with
Pierce Coomassie brilliant blue protein assay. Protein samples (30–60
µg) were resolved on a 12% SDS-polyacrylamide gel electrophoresis
(PAGE) and transferred to Immobilon polyvinylidene difluoride (PVDF)
membranes (Millipore Corporation, Bedford, MA). Western blots were
performed using Arf (1/2000), RhoA (1/1000), or PKC (1/1000)
antibodies, and proteins were revealed using the enhanced
chemiluminescence (ECL) detection system as described previously
[15
].
Statistics
Data are expressed as means ± SE. Data were
analyzed using the Student’s paired t-test (two-tailed) or
the Mann-Whitney test (nonparametric test) to determine the level of
significance between the treated samples and the appropriate controls
(*P<0.05).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 View larger version (16K): [in a new window] |
Figure 1. Effect of Ro20-1724 on CGS21680-mediated inhibition and CSC-induced
increase in PLD activity. Labeled neutrophils were prewarmed at 37°C
for 5 min. (A) Where indicated, the cell suspensions were pretreated
for an additional 5 min with 0.1 U/mL ADA, 1 µmol/L CSC, 10 µmol/L
Ro20-1724, or 1 µmol/L CGS21680, with or without Ro20-1724, in the
absence ( ) or the presence ( ) of 10 µmol/L CB. (B) The samples
were pretreated with 10 µmol/L CB and incubated for 5 min in the
presence of 1 µmol/L CSC or 10 µmol/L Ro20-1724 alone or in
combination. Cells were stimulated with 0.1 µmol/L fMLP for 10 min in
the presence of 1% ethanol. PLD activity was measured as described in
Materials and Methods. The levels of [3H]PEt formed are
expressed as the percentage of the radioactivity in the total lipid
extracts. The data are the means ± SE of two sets of
independent experiments (n=3 for each set).
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[15
], we conducted experiments to assess whether
Ro20-1724 increased the ability of CGS21680 to suppress the recruitment
of these PLD1 activation cofactors to membranes. As shown in
Figure 2
, Arf1 and RhoA were recruited to membranes in response to
stimulation with fMLP. A small increase in the amount of
membrane-associated PKC was also dectected when the blots were
overexposed (not shown; see also [15
]). As shown for PLD
activity, the removal of extracellular adenosine by ADA (lane 3) or the
addition of the A2a receptor antagonist CSC (lane 5) markedly increased
the fMLP-induced recruitment of PKC, RhoA, and Arf1 to membranes. In
the absence of ADA, CGS21680 (lane 4) or Ro20-1724 (lane 6) alone
decreased slightly the membrane recruitment of RhoA and Arf1 induced by
fMLP, but their combination reduced the levels of membrane-associated
RhoA and Arf to basal levels (lane 7). As estimated by densitometric
analyses of the blots (not shown) and illustrated (see Fig. 4
), the
stimulatory effect of the A2a receptor antagonist CSC on the
translocation of PLD1 activation factors was not attenuated
significantly by Ro20-1724 (lane 8).
 View larger version (51K): [in a new window] |
Figure 2. Effect of Ro20-1724 on CGS21680-mediated inhibition and CSC-mediated
increase of fMLP-induced translocation of PKC, RhoA, and Arf1 to
membranes. Neutrophils were prewarmed at 37°C for 5 min. Where
indicated, the cell suspensions were treated with 10 µmol/L CB in the
presence (lane 3) or the absence (lanes 1, 2, and 4–8) of 0.1 U/mL
ADA; 10 µmol/L Ro20-1724 (lanes 6–8); 1 µmol/L CGS21680, with
(lane 7) or without (lane 4) Ro20-1724; or 1 µmol/L CSC, with (lane
8) or without (lane 5) Ro20-1724 for 5 min. Neutrophils were stimulated
with 0.1 µmol/L fMLP for 2 min (lanes 2–8) and neutrophil membranes
were prepared as described in Materials and Methods. The samples were
probed for PKC, RhoA, and Arf1. The data are from one experiment
representative of three similar experiments.
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 View larger version (44K): [in a new window] |
Figure 4. Effect of cAMP-elevating agents on fMLP-mediated translocation of
PKC, RhoA, and Arf1 to membranes. Neutrophils were prewarmed at
37°C for 5 min and incubated with 10 µmol/L Ro20-1724 (lanes 2, 4,
6, and 8) or 10 µmol/L forskolin (lanes 3, 4, 7, and 8), alone or in
combination (lanes 4 and 8), for 25 min at 37°C. Cells suspensions
were incubated with 10 µmol/L CB and in the presence of 0.1 U/ml ADA
(lanes 1–8) for an additional 5 min prior to stimulation with 0.1
µmol/L fMLP for 2 min (lanes 5–8). Reactions were stopped, and
neutrophil membranes were prepared as described in Materials and
Methods. The samples were probed for PKC, RhoA, and Arf1. The data
are from one experiment representative of three similar experiments.
Quantitation by densitometry of Arf and RhoA in neutrophil membranes is
shown in the lower left and right panels, respectively
(n=3). *, P < .05 using the Student’s
paired t-test.
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 View larger version (42K): [in a new window] |
Figure 5. Effect of 2',5'-ddADO on CGS21680 and isoproterenol-mediated inhibition
of fMLP-induced PLD activity. [3H]-Labeled neutrophils
were prewarmed at 37°C for 5 min and incubated with the indicated
concentrations of 2',5'-ddADO for 5 min at 37°C. Cells were
stimulated with 0.1 µmol/L fMLP for 10 min in the presence of 1%
ethanol. PLD activity was measured as described in Materials and
Methods. The data are expressed as a percentage of the levels of
[3H]PEt formed in fMLP-stimulated cells in the presence
of CB. * and , P < .05 as compared with
fMLP-stimulated neutrophils in the absence or the presence of CGS21680,
respectively, using the Mann-Whitney test. (A) Neutrophils were treated
with 10 µmol/L CB (no ADA) and incubated with or without 1 µmol/L
CGS21680 for an additional 5 min prior to stimulation. The data are
means ± SE of six independent experiments, except for
the highest concentration of 2',5'-ddADO (n=2). (B)
Neutrophils were treated with 10 µmol/L CB (no ADA) and incubated
with or without 1 µmol/L isoproterenol for an additional 5 min prior
to stimulation. The data are means ± SE of six
independent experiments, except for the highest concentration of
2',5'-ddADO (n=2). (C) Neutrophils were treated with 10
µmol/L CB (no ADA) and incubated with or without 0.1 µmol/L
CGS21680 for an additional 5 min prior to stimulation. The data are
means ± SE of three independent experiments, each
performed in duplicate. (D) Neutrophils were treated with 10 µmol/L
CB and 0.1 U/ml ADA and incubated with or without 0.1 µmol/L CGS21680
for an additional 5 min prior to stimulation. The data are means ± SE of three independent experiments, each performed in
duplicate.
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Figure 3. Effect of ADA, CGS21680, and CSC on fMLP-induced cAMP formation.
Neutrophils were prewarmed at 37°C for 5 min and treated with 10
µmol/L Ro20-1724 for 25 min at 37°C. (A) Cell suspensions were
incubated with 10 µmol/L CB in the absence ( ) or the presence
(•) of ADA (0.1 U/mL ADA) for an additional 5 min and stimulated with
0.1 µmol/L fMLP (arrow). (B) Neutrophils were incubated with 10
µmol/L CB and 1 µmol/L CGS21680 in the absence () or the
presence (•) of ADA (0.1 U/mL ADA) for an additional 5 min prior to
stimulation with 0.1 µmol/L fMLP (arrow). (C ) Cell suspensions were
incubated with 10 µmol/L CB (no ADA) in the absence () or the
presence (•) of 1 µmol/L CSC for an additional 5 min prior to
stimulation with 0.1 µmol/L fMLP (arrow). At selected times, the
reactions were stopped. cAMP was extracted and measured as described in
Materials and Methods. The data are means ± SE of
three independent sets of experiments, each performed in duplicate
(n=3 for each set). *, P < .05 using the
Student’s paired t-test.
|
Effects of Ro20-1724 and forskolin on stimulation of PLD activity
induced by fMLP in ADA-treated neutrophils
Adenosine accumulates in a time-dependent manner in neutrophil
suspensions [15
] and modulates the kinetics of the
accumulation of cAMP in response to stimulation with fMLP (Fig. 3)
. To
further establish that cAMP elevation reduces fMLP-induced activation
of PLD, we investigated the effect of Ro20-1724 and forskolin on
fMLP-induced PLD activation in ADA-treated cells. The PDE inhibitor,
which increased basal and fMLP-induced cAMP elevation (Fig. 3)
, reduced
the stimulatory effects of fMLP on the levels of PEt formation in a
time- and concentration-dependent manner. A preincubation of the cells
with 10 and 100 µM Ro20-1724 for 5, 15, 30, and 60 min prior to
stimulation with fMLP reduced the levels of PEt formed by 15.5 ±
3.6%, 31 ± 8%, 47.3 ± 8%, and 51.2 ± 3.6%
(n=3) and by 32.3 ± 2.5%, 47.4 ± 2.4%,
57.5 ± 5%, and 65.3 ± 5.4% (n=3),
respectively. Forskolin also inhibited fMLP-induced PLD activation in a
time- and dose-dependent manner (unpublished results). The suppressive
effects of Ro20-1724 and forskolin on PLD activity were additive.
Indeed, a preincubation of cell suspensions with 10 µM forskolin and
Ro20-1724 alone or in combination for 30 min reduced the levels of
[3H]PEt formed by 20 ± 5.9%, 17 ± 2.5%, and
38.7 ± 6.6%, respectively. The addition of a suboptimal
inhibitory concentration (1 nM) of the A2a receptor agonist CGS21680
reduced the amounts of PEt formed by 22 ± 5.6% (n=3).
In the presence of CGS21680, the inhibitory effects of Ro20-1724 and
forskolin alone or in combination were enhanced to 43 ± 4.8%,
43 ± 9.4%, and 58 ± 5.7%, respectively. We next
investigated whether the fMLP-mediated translocation of PKC, RhoA,
and Arf1 to membranes was modulated by Ro20-1724 and forskolin
(Fig. 4
). The basal levels of RhoA, recovered in membranes obtained from
ADA-treated but otherwise unstimulated neutrophils (lane 1), were
slightly reduced by a pretreatment of the cells with Ro20-1724 (lane 2)
alone or in combination with forskolin (lane 4). The recruitment of
PKC, RhoA, and Arf1 induced by fMLP (lane 5) was not reduced
significantly by Ro20-1724 (lane 6) or forskolin (lane 7). As observed
for PLD activity, the inhibitory effects of Ro20-1724 and forskolin on
the fMLP-mediated translocation of PKC and small GTPases to
membranes were additive (Fig. 4
, lane 8). Together, the data support
the hypothesis that cAMP acts as the second messenger for inhibition of
PLD activation and recruitment of PLD cofactors to membranes.
Effect of 2',5'-dideoxyadenosine (2',5'-ddADO) on CGS21680 and
isoproterenol-mediated inhibition of fMLP-mediated activation of PLD
If cAMP is the second messenger for adenosine A2a
receptor-mediated suppression of PLD activity, then inhibition of
adenylyl cyclase should reduce the inhibitory effects of A2a receptor
agonists or other receptors that stimulate the accumulation of cAMP. To
determine whether inhibition of PLD activity by adenosine A2a receptor
is mediated by cAMP, we studied the effect of 2',5'-ddADO, a ligand for
the P-site on adenylyl cyclase, which inhibits its catalytic activity
[32
]. In these experiments, neutrophils were not treated
with ADA to allow accumulation of endogenous adenosine. As shown in
Figure 5
, the addition of 100, 300, and 900 µM 2',5'-ddADO to neutrophils
enhanced fMLP-induced PLD activity by 44 ± 11% (n=6),
85 ± 17% (n=6), and 89 ± 36% (n=2),
respectively. The increases in PEt formation were similar to those
induced by the addition of ADA to cell suspensions or by blocking A2a
receptors using selective antagonists prior to stimulation with fMLP
(Fig. 1)
. As demonstrated previously [15
], 1 µmol/L
CGS21680 reduced the amounts of [3H]PEt formed to
40.3 ± 3.1% (n=6) of the fMLP-induced response (Fig. 5A)
. A treatment with 2',5'-ddADO (100, 300, and 900 µM) partially
reversed the suppressive effects of CGS21680, increasing the amounts of
[3H]PEt formed to 45.9 ± 3.5% (n=6),
63.5 ± 7.8% (n=6), and 58.8 ± 12.3%
(n=2) of the fMLP-induced response, respectively. Other
cAMP-elevating agonists also reduced fMLP-induced PLD activation.
Figure 5B
shows that stimulation of ß2-adrenoreceptors with
isoproterenol also reduced fMLP-induced PLD activity by 45.1 ±
2.8%. 2',5'-ddADO reversed the inhibitory effect of 1 µmol/L
isoproterenol (Fig. 5B)
more efficiently than those of 1 and 0.1
µmol/L CGS21680 (Fig. 5A
and 5C
, respectively). When neutrophil
suspensions were pretreated with ADA, 2',5'-ddADO has no noticeable
effect on fMLP-mediated [3H]PEt formation (Fig. 5D)
.
However, 2',5'-ddADO (100, 300, and 900 µM) was able to partially
reverse the suppressive effects of 0.1 µmol/L CGS21680 on PLD
activity. These increases of [3H]PEt accumulation were
statistically significant, but the levels formed in response to fMLP
were twofold lower than those obtained without any CGS21680 (Fig. 5D)
.
These data further demonstrated that adenosine A2a receptors reduced,
at least in part, PLD activation by a cAMP-dependent mechanism.
Effect of modulators of PKA activity, Sp-cAMPs-AM and Rp-cAMPs-AM,
on fMLP-induced PLD activity and translocation of PKC, RhoA, and
Arf1 to membranes
The above results suggested that the sustained elevation of cAMP
resulting from the activation of adenylyl cyclase by engagement of A2a
receptors reduced the ability of fMLP to turn on the PLD signaling
pathway. To determine whether adenosine inhibited the stimulation of
PLD activity through the activation of PKA, neutrophils were incubated
with the PDE-resistant and cell-permeable activators and inhibitors of
PKA [33
], Sp-cAMPs-AM and Rp-cAMPs-AM, respectively. In
these experiments, neutrophils were preincubated with the cAMP
analogues for 30 min to allow their uptake, their hydrolysis by
intracellular esterases, and accumulation inside the cells. Rp-cAMPs-AM
was without significant effect on the levels of [3H]PEt
formed when neutrophils were treated with ADA prior to stimulation with
fMLP (Fig. 6A
). Concentrations of Rp-cAMPs-AM >50 µM were not tested because
they produced a small inhibition of PLD activity (not shown). In
contrast, when ADA was omitted, 10 and 50 µM Rp-cAMPs-AM enhanced by
1.18 ± 0.15- and 1.66 ± 0.2-fold, respectively, the
stimulation of PLD activity induced by fMLP. The stimulatory effects of
the PKA inhibitor on PLD activation were similar to those shown for ADA
(Fig. 1)
, the A2a receptor antagonist CSC (Fig. 1)
and the P-site
inhibitor of adenylyl cyclase 2',5'-ddADO (Fig. 6A
and 6B)
. In
contrast, Sp-cAMPs-AM reduced fMLP-induced PLD activity in a
concentration-dependent manner (Fig. 6B)
. Inhibition of fMLP-mediated
[3H]PEt formation by Sp-cAMPs-AM was larger when
neutrophils were pretreated with ADA to remove extracellular adenosine,
thereby suggesting that PKA was already activated by endogenous
adenosine.
 View larger version (18K): [in a new window] |
Figure 6. Effect of Sp-cAMPs-AM and Rp-cAMPs-AM on fMLP-induced PLD activity.
[3H]-Labeled neutrophils were prewarmed at 37°C for 5
min and incubated for 30 min with the indicated concentrations of
Rp-cAMPs-AM (A) or Sp-cAMPs-AM (B). Neutrophils were treated
with 10 µmol/L CB and incubated for an additional 5 min with or
without 0.1 U/ml ADA prior to stimulation with 0.1 µmol/L fMLP for 10
min in the presence of 1% ethanol. PLD activity was measured as
described in Materials and Methods. The levels of [3H]PEt
formed are expressed as a percentage of the fMLP-induced response. The
data are means ± SE of a minimum of four independent
experiments. *, P < .05 when compared with the control
with no Rp-cAMPs-AM or Sp-cAMPs-AM.
|
, RhoA, and Arf1 to neutrophil membranes, we investigated
whether translocation of PLD1 cofactors to membranes was also regulated
by PKA using Sp-cAMPs-AM and Rp-cAMPs-AM. Densitometric analyses of the
blots indicate that a preincubation of cell suspensions with the
inhibitor of PKA, Rp-cAMPs-AM, in the absence of ADA enhanced
fMLP-induced recruitment of Arf1 and RhoA to neutrophil membranes
(Fig. 7A
). In contrast, the addition of the PKA activator, Sp-cAMPs-AM,
and ADA consistently reduced fMLP-mediated translocation of RhoA and
Arf1 to membranes (Fig. 7B)
. Together, these data suggest that PKA
inhibits fMLP-induced PLD activation and PLD-cofactor recruitment.
 View larger version (29K): [in a new window] |
Figure 7. Effect of Sp-cAMPs-AM and Rp-cAMPs-AM on fMLP-induced translocation of
PKC, RhoA, and Arf1 to membranes. Neutrophils were prewarmed at
37°C for 5 min and incubated for 30 min with the indicated
concentrations of Rp-cAMPs-AM (A) or Sp-cAMPs-AM (B). Neutrophils were
incubated for an additional 5 min with 10 µmol/L CB in the absence
(A) or the presence (B) of 0.1 U/ml ADA prior to stimulation with 0.1
µmol/L fMLP for 2 min at 37°C. Reactions were stopped, and
neutrophil membranes were prepared as described in Materials and
Methods. The samples were probed for PKC, RhoA, and Arf1. The data
are from one experiment representative of four similar experiments.
Quantitation by densitometry of Arf and RhoA in neutrophil membranes is
shown in the lower left and right panels, respectively
(n=4). *, P < .05 using the Student’s
paired t-test.
|
, RhoA, and Arf1 to membranes and small
GTPases. As estimated by densitometric analyses of the blots, H89 alone
slightly increased the basal amounts of PKC, RhoA, and Arf1
associated with membranes and enhanced their translocation in response
to stimulation with fMLP (Fig. 8B
, lane 4). Furthemore, H89 totally
reversed the inhibitory effect of CGS21680 on fMLP-induced
translocation of PKC, RhoA, and Arf1 to neutrophil membranes (Fig. 8B
, lanes 5 and 6). Together, the data are consistent with the
hypothesis that the inhibition of the stimulation of PLD activity and
of the recruitment of small GTPases and PKC to membranes induced by
fMLP observed upon A2a receptor occupancy is mediated by an activation
of PKA (Fig. 9
).
 View larger version (29K): [in a new window] |
Figure 8. Effect of H89 on fMLP-mediated PLD activity and translocation of
PKC, RhoA, and Arf1 to membranes. (A) [3H]-Labeled
neutrophils were prewarmed at 37°C for 5 min. Where indicated, the
samples were treated with 10 µmol/L CB, 20 µmol/L H89, and 0.1
µmol/L CGS21680 in the absence () or the presence () of 0.1
U/ml ADA and incubated for an additional 5 min prior to stimulation
with 0.1 µmol/L fMLP for 10 min at 37°C in the presence of 1%
ethanol. PLD activity was measured as described in Materials and
Methods. The levels of [3H]PEt formed are expressed as a
percentage of the radioactivity in the total lipid extracts. The data
are means ± SE of three independent experiments. *
and , P < .05 as compared with fMLP-stimulated
neutrophils in the absence or the presence of ADA, respectively, using
the Student’s paired t-test. (B) Neutrophils were processed
as in A and incubated in the absence of 0.1 U/ml ADA for 5 min prior to
stimulation with fMLP for 2 min. Reactions were stopped, and neutrophil
membranes were prepared as described in Materials and Methods. The
samples were probed for PKC, RhoA, and Arf1. The data are from one
experiment representative of three similar experiments. Quantitation by
densitometry of Arf and RhoA in neutrophil membranes is shown in the
lower left and right panels, respectively (n=3).
|
 View larger version (28K): [in a new window] |
Figure 9. Model of PLD regulation by adenosine A2a receptors. Occupancy of
adenosine A2a receptors (A2a-R) by adenosine or the agonist CGS21680
enhances adenylyl cyclase activation; then, cAMP activates PKA. PKA
activation appears to inhibit fMLP-induced translocation of PKC and
of small GTPases, thereby reducing fMLP-induced PLD activation. PKA
activity can be enhanced indirectly by stimulating adenylyl cyclase
with forskolin, inhibiting the degradation of intracellular cAMP by PDE
IV with Ro20-1724, or directly by the PKA activator Sp-cAMPs. The
removal of extracellular adenosine with ADA or the addition of the
adenosine A2a receptor antagonist CSC reduces cAMP accumulation and PKA
activation. PKA activity is also reduced by the inhibition of adenylyl
cyclase with 2',5'-ddADO or by the PKA inhibitors H89 and Rp-cAMPs.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenosine exerts its biological effects via the pharmacologically distinct receptor subtypes, A1, A2a, A2b, and A3 [35 , 36 ]. The expression of A2a receptors on neutrophils is supported by the detection of its mRNA by reverse transcriptase polymerase chain reaction [37 ]. Furthermore, pharmacological and immunological evidence has identified functional A1 and A2 receptors on neutrophil plasma membranes [38 , 39 ]. Endogenous adenosine is a potent autocrine regulator of PLD activity in chemoattractant-activated neutrophils, and our previous pharmacological studies support the involvement of A2a receptors in the inhibition of fMLP-stimulated PEt formation [15 ]. As initially described by Jackowski and Sha’afi [40 ], fMLP increases cAMP levels in neutrophils. This elevation in the levels of cAMP can be divided in two distinct phases, one short-lived and another more sustained. In the present study, we demonstrated that the removal of endogenously produced adenosine by ADA or the addition of the selective A2a receptor antagonist CSC inhibited the sustained phase of cAMP accumulation without affecting the initial transient elevation in cAMP levels. In contrast, the addition of the selective A2a receptor agonist CGS21680 to ADA-treated neutrophils restored the long-lasting cAMP elevation. The persistent increases in cAMP levels were functionally correlated to the suppression of fMLP-induced PLD activity and highlight the role of A2a receptors in regulating the long-lasting increase in cAMP levels. This is consistent with previous studies showing that intracellular cAMP elevation as the result of ligand-induced activation or neutrophil treatment with cell membrane-permeable cAMP analogues reduced fMLP-stimulated PLD activity [22 , 23 ].
Although the occupancy of A2a receptors has been suggested to uncouple
chemoattractant receptors from their downstream signal transduction
pathways [25
], adenosine does not interfere with
PLC-mediated inositol 1,4,5-triphosphate formation [20
],
the mobilization of intracellular Ca2+, or the
early, transient increases in diacylglycerol formation induced by fMLP
[21
, 41
]. However, adenosine has been shown
to inhibit the influx of Ca2+ induced by fMLP
[42
] or platelet-activating factor (PAF)
[14
] and to diminish the late and sustained increases in
diacylglycerol that follow fMLP stimulation [41
]. We
attributed this late and persistent increase in diacylglycerol
formation to the sequential actions of a PLD and of lipid
phosphohydrolases [15
, 43
]. Therefore, the
inhibitory effects of adenosine on the late phase of accumulation of
diacylglycerol are consistent with the results of the present and
previous studies [15
] describing inhibitory effects of
adenosine on the stimulation of the activity of PLD in human
neutrophils. A2a receptors are linked to Gs [44
,
45
], and their stimulation by selective agonists
stimulates cAMP formation in intact granulocytes [21
,
27
] or in neutrophil membranes [38
]. The
ability of adenosine to induce long-lasting increases in the levels of
cAMP in response to fMLP stimulation has been demonstrated only in the
presence of nonmethylxanthine inhibitors of PDE [26
] but
not with the xanthine-based PDE inhibitors, 3-isobutyl-1-methylxanthine
[46
] or theophylline [26
], which are also
nonselective adenosine receptor antagonists [19
].
Furthermore, stimulation of ß-adrenergic receptors, which belong to
Gs-coupled receptors and are known to increase cAMP levels in
neutrophils, reduced fMLP-induced PLD activity [26
,
46
]. These suppressive effects of A2a and ß adrenergic
receptors are likely mediated through activation of adenylyl cyclase,
because 2',5'-ddADO, an inhibitor of adenylyl cyclase
[33
], reversed, at least in part, the suppressive effect
of A2a and ß-adrenergic receptor agonists.
Depending on the cell types studied, increases in cAMP inhibit
[47
48
49
] or stimulate [50
,
51
] PLD activity via activation of PKA. PLD1a is the
major PLD isoform in human neutrophils [52
]. PLD1
requires phosphatidylinositol 4,5-bisphosphate for activity
[53
] and is activated by Arf, RhoA, and PKC in vitro
[54
]. These cytosolic proteins are recruited to
membranes of fMLP-stimulated cells, and there is evidence suggesting
that they mediate fMLP-induced PLD activity in human granulocytes
[30
, 55
]. Furthermore, the engagement of
A2a receptors by selective agonists reduces PLD activity by limiting
the recruitment of Arf, RhoA, and PKC to membranes
[15
]. As summarized in Figure 9
, the cAMP-PKA pathway is
likely to play a key role, because the blockade of PKA activity by H89
or Rp-cAMP increased, whereas the PKA activator, Sp-cAMP, reduced PEt
formation and the levels of small GTPases and PKC associated with
membranes. It is interesting that elevations of intracellular cAMP
concentrations have been shown to inhibit chemoattractant-induced
activation of RhoA [56
]. There is evidence that PKA
phosphorylates RhoA [57
] and that the phosphorylation by
PKA of RhoA associated to granulocyte membranes reduced GTP
S-induced
PLD activity [58
]. However, besides the phosphorylation
of RhoA, the inhibition of fMLP-induced Arf recruitment involves other
signaling molecules because PKA has no effect on Arf-mediated
activation of PLD in isolated neutrophil membranes [58
].
Moreover, PKA activity has been shown to promote the association of
Arf1 to Golgi membranes [59
]. Our data do not extend
this concept to neutrophils because PKA activation correlated with the
suppression of Arf1 recruitment to membranes. The molecular mechanisms
implicated in the cAMP/PKA-dependent inhibition of Arf1 activation or
translocation to neutrophil membranes remain to be clarified.
Although adenosine increases cAMP levels, A2a receptors have also been shown to initiate some functional responses via cAMP-independent pathways in human neutrophils [3 ]. Accordingly, the inhibition of PKA by KT-5720 failed to reverse the inhibition of apoptosis [60 ] of fMLP-mediated superoxide-anion generation [25 ] or the activation of serine/threonine phosphatases [29 ] by A2 receptor agonists. In contrast, inhibition of PKA by H89 or Rp-cAMP has been shown to suppress the inhibitory effect of A2 receptor agonists on actin polymerization and phagocytosis [5 ], fMLP-induced activation of the respiratory burst [37 , 61 ], and PAF-induced leukotriene biosynthesis and translocation of 5-lipoxygenase [62 ]. The activation of PLD correlates with activation of the NADPH oxidase [24 , 63 , 64 ]. A model in which a phosphatidic acid (PA)-activated kinase is involved in the activation of the NADPH oxidase has been proposed [24 ]. Elevation of intracellular cAMP by physiological agonists (e.g., PGE2, histamine, or ß-adrenergic agonists) inhibits the respiratory burst and PLD activation in fMLP-stimulated neutrophils, consistent with a functional link [22 , 23 ]. The inhibition of PLD activation and superoxide anion generation by adenosine A2a receptor activation is consistent with a role for PA-derived PLD in NADPH activation [15 , 37 , 61 ]. However, determining the impact of adenosine A2a receptor occupancy on the activation of rac, a small GTPase required for NADPH oxidase activity [65 ], is required to critically evaluate the role of PA-derived PLD in this model.
In summary, the results of this study demonstrate that endogenous
adenosine inhibits the activation of PLD through the engagement of A2a
receptors, which changes the transient elevations of cAMP induced by
fMLP into persistent elevation of cAMP levels. Our data also indicate
that the inhibition of fMLP-induced PLD activity is functionally linked
to inhibition, via a PKA-regulated mechanism, of fMLP-induced
recruitment of Arf, RhoA, and PKC to membranes. Our studies are
consistent with the hypothesis that adenosine uncouples chemoattractant
receptors from the PKC and small G protein-dependent activation of PLD1
activation via PKA-dependent mechanisms. These various observations are
schematically depicted in the model shown in Figure 9
.
Received June 14, 2001; revised September 19, 2001; accepted September 24, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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