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Introduction
Cholecystokinin (CCK) is a gut-brain peptide that exerts
a variety of physiological actions in the gastrointestinal tract
and central nervous system (CNS) through activating cell
surface CCK receptors[1]. CCK receptors have been
pharmacologically classified into 2 subtypes, CCK-A receptors
(CCK-AR) and CCK-B receptors (CCK-BR), according to their
affinity for the peptide agonists CCK and
gastrin[2]. Our previous experiments demonstrated that CCK-8, as an
intestinal neuropeptide, not only protected gastric mucosa against
alcohol-induced injury, but also was a potent protective
agent against acute lung injury caused by
lipopolysaccharide (LPS)[3,4]. It reduced pulmonary artery hypertension
(PAH) and lessened inflammatory lesions in lung tissues of
endotoxin shock (ES) rats[3,4]. Studies from our laboratory
show that sulfated CCK-8 (sCCK-8) can inhibit LPS-induced
TNF-a production in vitro, sCD14 release and mCD14
expression in rat pulmonary interstitial macrophages
(PIM)[5]. The production of proinflammatory cytokines, including
TNF-a, IL-1b, and IL-6 in ES rats, was also inhibited by sCCK-8
in vivo[6,7]. The above data suggest that sCCK-8 has an
anti-inflammatory effect, confirmed by a morphological
observation that sCCK-8 clearly lessened the inflammatory lesions
in the spleen and the liver tissues in endotoxin shock
rats[4]. However, the anti-inflammatory mechanisms and signaling
pathways activated by CCK-8 remain unclear.
Several signaling pathways are involved in the
activation of monocytes/macrophages induced by LPS, including
the mitogen-activated protein kinase (MAPK) and the
protein kinase C (PKC) pathway, and there is a lot of cross-talk
between them. PKC, as an important message molecule,
participates extensively in immune regulation and inflammatory
signal transduction. In particular, PKCz plays a pivotal role
in the complicated signaling networks induced by
LPS[8,9]. Several studies show that LPS-induced activation of the
extracellular signal-regulated kinases (ERK) in alveolar
macrophages is mediated by sequential activation of
phosphatidylcholine-specific phospholipase C (PC-PLC), induction of
cellular diacyglycerol (DAG) and ceramide, and activation of
PKCz[10]. Previously we reported that CCK-AR and CCK-BR
mRNA expressions were detected by reverse
transcription-polymerase chain reaction. The presence of functional CCK
receptors was confirmed by radioligand binding assay in rat
PIM, and their expressions were upregulated by
LPS[11]. Increasing evidence supports the concept that the signal
transduction cascades mediated by CCK receptors are cell specific.
For example, CCK was found to activate cAMP and PKC
regulated signaling pathways in pancreatic acinar cells, but
CCK-8 induced a significant decrease in membrane and
cytosol PKC activity in murine lymphocytes, neutrophils, and
peritoneal macrophages, as well as an increase of
intracellular cAMP levels[12-14]. As PIM plays an important role in the
inflammatory response to LPS in the lungs and little is known
about the effect of CCK-8 on DAG-PKC signaling pathway
in PIM, the present study was undertaken to investigate the
effect of CCK-8 on DAG contents, PKC activities, and
PKCz translocation in resting and LPS-stimulated rat PIM, and to
explore the anti-inflammatory molecular mechanisms
activated by CCK-8.
Materials and methods
Chemicals and reagents Collagenase IA, LPS
(Escheri-chia coli 0111:B4), CCK-8s, proglumide, CR-1409, CR-2945,
aprotinin, leupeptin, and DNaseI were obtained from Sigma
Chemical Co. RPMI-1640 culture medium,
phenylmethyl-sulfonyl-fluoride (PMSF), and nitrocellulose membranes were
obtained from Gibco BRL. DAG biotrak assay reagents
system (RPN 200) and Amprep Si minicolumns (RPN 1906) were
obtained from Amersham Biosciences. SignaTECT® PKC
assay system and Gel shift assay system were obtained from
Promega. Diethylaminoethyl cellulose (DE52) was obtained
from Whatman BioSystems. Affinity purified polyclonal
nPKCz antibodies (C-20), peroxidase-conjugated goat
anti-rabbit IgG, and Western blotting luminol reagent (sc-2048)
were obtained from Santa Cruz Biotechnology. All other
reagents were of analytic pure grade.
Animals Adult healthy female Sprague-Dawley (SD) rats
(180-220 g, Grade II, Certificate No 04057) were obtained from
the Experimental Animal Center of Hebei Province (Shijiazhuang, China).
Cell culture and treatment PIM were isolated from
perfused rat lungs with a collagenase digestion technique,
modified as Wizemann et al[15]
. PIM were harvested with PBS containing 0.4 g/L edetic acid, pelleted by centrifugation
(4 ºC, 400×g, 10 min) and resuspended with serum-free
medium. Cells were incubated with CCK-8 at different
concentrations or stimulated with LPS (10 mg/L) in the absence
or presence of CCK-8, proglumide, CR-1409, and CR-2945 for
1 h. At the end of incubation, PIM were centrifuged at
400×g for 60 s, and the pellets were measured for DAG contents,
PKC activities, and translocation.
Measurement of intracellular DAG
contents Total cell lipids were extracted using a modification of the method of
Bligh and Dyer[16]. Intracellular DAG contents were
measured using the DAG assay reagents system from Amersham
Biosciences. A radioenzymatic assay uses the diacylglycerol
kinase and defined mixed micelle conditions to solubilize the
DAG present and allow its quantitative conversion to
[32P]phosphatidic acid in the presence of
[g-32P]ATP. Following the enzyme-catalyzed phosphorylation of DAG, the
[32P]phosphatidic acid reaction product was extracted and
separated by Amprep chromatography. The radioactivity
attributable to [32P]phosphatidic acid was determined by
liquid scintillation counting. The amount of DAG present in
the sample was calculated from the amount of
[32P]phosphatidic acid produced and the specific activity of the ATP
according to the standard curve was expressed as pmol per
2×106 cells.
Preparation of cytosol and membrane
fractions After treatment, the PIM were washed with 5 mL ice-cold PBS and
centrifuged at 250×g for 60 s. The supernatant was discarded
and the cells were suspended in iced 0.5 mL of extraction
buffer [Tris-HCl 25 mmol/L (pH 7.4) containing edetic acid
0.5 mmol/L, egtazic acid 0.5 mmol/L, b-mercaptoethanol
10 mmol/L, leupeptin 1 mg/L, and aprotinin 1 mg/L] and
sonicated on ice with 10-s bursts, each preceded by a 10-s
pause. The homogenates were centrifuged at
100 000×g for 60 min at 4 ºC and the supernatant was used for assay of
PKC (cytosol fraction). The pellet (representing the
membrane fraction) was suspended in 2 mL of iced extraction
buffer containing 0.05% Triton X-100, shaken for 60 min at 4
ºC and centrifuged at
100 000×g at 4 ºC for 60 min. The
supernatant was taken for assay of PKC (membrane fraction).
Cytosol and membrane fractions were partially purified
using diethylaminoethyl (DEAE)-cellulose chromatography.
The samples were passed over individual 1-mL columns
pre-equilibrated with extraction buffer. Following washing the
columns 1 time with 5 mL of extraction buffer, the fractions
of PKC were eluted with 5 mL of extraction buffer containing
NaCl 200 mmol/L, stored at 4 ºC, and generally assayed within
24 h.
PKC activity assay PKC activity was analyzed by
measuring the incorporation of 32P from
[g-32P]ATP (185 PBq/mol) into peptide Neurogranin
(28AAKIQAS*-FRGHMA-RKK43), a specific substrate of PKC. The standard assay mixture
(25 μL) contained extraction buffer 5 μL, 5 μL PKC
co-activation buffer (5×), 5 μL PKC substrate peptide 0.5 mmol/L,
and 5 μL of ATP mixture containing ATP 0.5 mmol/L and
[g-32P]ATP 18. 5 GBq (0.5 Ci). Control reaction used a
control buffer instead of PKC substrate peptide. The reaction
was terminated by adding ice-cold 7.5 mol/L guanidine
hydrochloride 12.5 μL at 30 ºC after 5 min. Each terminated
reaction mixture (10 μL) was then spotted onto an SAM
membrane. The membrane was rinsed with 2 mol/L NaCl and
2 mol/L NaCl in 1% phosphoric acid and deionized water,
respectively. Aliquots of 5 μL in any 2 reactions were
spotted onto SAM membrane. All membranes were dried at room
temperature and put into liquid scintillator. Radioactivity
was counted. All values represented the mean from 3
separate culture dishes, with an average difference between
duplicates of <±5%. Proteins were measured using Bradford¡¯s
method by using bovine serum albumin as a standard.
Immunoblot analysis The effect of CCK-8 on
distribution and on LPS-induced translocation of
PKCz was determined using quantitative immunoblot analysis. Crude
cytosolic and membranous fractions from PIM were prepared as
described above, except that DE52 column chromatography
was omitted. Protein concentrations of fraction were
measured using Bradford¡¯s method using bovine serum albumin
as standard. Aliquots of sample eluates were added to sample
preparation buffer [Tris-HCl 0.05 mol/L, pH 6.8, 20% glycerol,
2% sodium dodecylsulfate (SDS), edetic acid 5 mmol/L,
egtazic acid 5 mmol/L, b-mercaptoethanol 10 mmol/L,
leupeptin 1 mg/L, aprotinin 1 mg/L, PMSF 0.5 mmol/L, and
0.1% bromphenol blue] and boiled for 5 min. The proteins
were separated by SDS-polyacrylamide (10% acrylamide) gel
electrophoresis and electrophoretically transferred to
nitrocellulose membranes. The completeness of transfer was
confirmed by staining the gel with Coomassie blue. The blots
were incubated at room temperature for 2 h with PBS
containing 5% nonfat dry milk and 0.05% Tween 20 (bovine
serum albumin/Tris buffer saline) to block non-specific sites.
The blots were washed with PBS, and incubated overnight
with affinity purified polyclonal nPKCz antibodies(1:200) at
4 ºC. The blots were washed 3 times for 15 min each with
PBS at room temperature and incubated at 37 ºC for 1 h with
peroxidase-conjugated goat anti-rabbit IgG antibody. The
blots were finally washed 3 times for 15 min each with Tris
buffer saline, incubated with enhanced chemiluminescence
detection reagents for 3-5 min and exposed to X-ray films
for up to 60 s. The density of the bands on the film was
analyzed by Gel-Pro analyzer version 3.1 software (Media
Cybernetics). The arbitrary unit
(Darea·Ddensity
) was used for expressing the relative level of
PKCz of cytosolic and membranous fractions from PIM.
Statistical analysis Data were expressed as mean±SD
and analyzed using analysis of variance and the least
significant difference test using an SPSS statistical program.
Statistical significance was accepted when
P<0.05. Half-maximal inhibition
(IC50) was calculated using the log-probit
method.
Results
DAG content For resting PIM, CCK-8 did not affect DAG
content at low concentrations
(1×10-12-1×10-7 mol/L,
P>0.05), but decreased it at high concentrations
(1×10-6-1×10-5
mol/L) compared with that of the control group
(P<0.01). Stimulation of PIM by LPS (10 mg/L) obviously increased DAG
content (P<0.01) (Figure 1). CCK-8 concentration-dependently
inhibited the LPS-induced DAG content, and a significant
inhibition was observed at high concentrations
(1×10-8-1×10-5 mol/L,
P<0.01), but not at low concentrations
(1×10-11-1×10-9 mol/L,
P>0.05) (Figure 1). The inhibitory effect of CCK-8 on DAG
content induced by LPS was abrogated in part by proglumide,
CR-1409, and CR-2945 in a concentration-dependent manner.
Proglumide was so potent that it had an obvious effect on
CCK-8-resultant inhibition of LPS-induced DAG content
increase at low concentrations, and even resulted in a full
reversal of it at high concentrations, and a similar effect was
found for CR-1409, but not for CR-2945, with CR-2945
showing a weak effect only at high concentrations. The
IC50 of proglumide, CR-1409, and CR-2945 were
(1.76±0.19)×10-6 mol/L,
(6.76±0.52)×10-5 mol/L, and
(5.45±0.42)×10-3 mol/L,
respectively (Figure 2).
PKC activity and translocation Treating resting PIM
with CCK-8, no changes of cytosolic and membrane-bound
PKC activities were found at low concentrations
(1×10-12-1×10-9 mol/L), but an increase of cytosolic PKC activities and
a decrease of membrane-bound PKC activities were observed
gradually with the increase of CCK-8 concentration, and
significant differences presented at
1×10-6-1×10-5 mol/L
(P<0.01) (Figure 3). LPS decreased cytosolic PKC activity
and increased membrane-bound PKC activity
(P<0.01), indicating that LPS could promote PKC translocation and change
its distribution, but did not affect its total activity in rat PIM.
Treating PIM with CCK-8 did not affect LPS-induced PKC
translocation at
1×10-11-1×10-10 mol/L, decreased it slightly
at 1×10-9-1×10-8 mol/L
(P<0.05), and inhibited it significantly
at 1×10-7-1×10-5 mol/L
(P<0.01), suggesting that CCK-8
inhibited LPS-induced PKC translocation in a
concentration-dependent manner (Figure 3). The inhibitory effect of
CCK-8 1×10-6 mol/L on LPS-induced PKC activity translocation in
rat PIM was attenuated by proglumide, CR-1409, and
CR-2945 at 1×10-4 mol/L, and their inverse rate (percentage of
CCK-8) were 58.9%, 48.1%, and 21.5%, respectively
(P<0.01) (Figure 4).
Immunoblot analysis For resting PIM, CCK-8 inhibited
PKCz translocation from cytosol to membrane only at high
concentrations (1×10-6 mol/L)
(P<0.05) (Figure 5). LPS promoted
PKCz translocation from cytosol to membrane (P<
0.01) and CCK-8 inhibited LPS-induced PKCz translocation
significantly at 1×10-8 and
1×10-6 mol/L (P<0.01, Figure 6).
The inhibitory effect of CCK-8 on LPS-induced PKCz
translocation was blocked by proglumide, CR-1409
(P<0.01), and CR-2945 (P<0.05) to some extent (Figure 7).
Discussion
CCK is a neuropeptide expressed in the endocrine I-cells
of the small intestinal mucosa and in widespread central and
peripheral neurons[17]. Whereas intestinal CCK regulates
the release of pancreatic enzymes and the contraction of the
gall bladder, neuronal CCK is a transmitter or modulator
assumed to be involved in a variety of CNS functions, such
as feeding behavior, anxiety, analgesia, memory,
immuno-modulation, and anti-opioid
effects[1,18,19]. CCK receptor is a G protein-coupled receptor with seven-transmembrane
domain that has attracted considerable interest as a target
for drug-discovery efforts, based on its important
physiological role in the gastric mucosa, CNS, and immune
cells[20]. CCK acting through its G protein-coupled receptor
is now known to activate a variety of intracellular signaling
mechanisms and, thereby, to regulate a complex array of
cellular functions in different types of cells. Data from several
labs indicates that CCK increases intracellular calcium in
human peripheral blood mononuclear cells and T
lymphocyte cell lines[21]. sCCK-8 can inhibit the mobility capacity
and the mitogen-induced lymphocytic proliferation, but can
increase the adherence and the spontaneouse proliferation
of lymphocytes[22,23]. In addition, sCCK-8 is a negative
modulator of several functions of resting murine peritoneal
macrophages and human neutrophils, including the production
of superoxide anion, phagocytosis and mobility, and the
inhibition of these activities is carried out through an
increase of intracellular cAMP levels and a decrease in PKC
activity[11]. In the present study sCCK-8 did not affect DAG
content, PKC activities, and PKCz translocation at low
concentrations, but decreased DAG content and inhibited
PKC activities and PKCz translocation at high
concentrations in rat resting PIM. Resting PIM were not sensitive to
the stimulation by sCCK-8, and CCK-8 was a negative
modulator of the DAG-PKC signaling pathway at physiological
concentrations, which was very important for maintaining
body homeostasis, and inhibited the DAG-PKC signaling
pathway at high con-centrations.
LPS is the major component of the outer leaflet of
Gram-negative bacteria and has profound immunostimulatory and
inflammatory capacity. LPS interacts with LPS-binding
protein, therefore allowing binding to CD14 and association
with the Toll-like receptor 4 (Tlr4) containing an intracellular
signaling domain. Binding of LPS to these receptors results
in the activation of several signaling cascades, such as PKC
and MAPK[24]. PKC isoforms are involved in a wide variety
of intracellular signaling events and play a significant role in
many aspects of immune response, from development,
differentiation, activation, and survival of lymphocytes to
macrophage activation[25]. PKCz is a member of the atypical
subfamily and has been widely implicated in the regulation
of cellular functions, particularly in the LPS-induced
inflammatory response[9]. In the present study, stimulation of PIM
by LPS increased DAG content, promoted PKC activities
and PKCz translocation, and changed its distribution, but
did not affect PKC total activity. In resting cells or in the
absence of lipid hydrolysis, PKC are localized primarily to
the cytosol. Translocation to the membrane from cytosol is
an important step in PKC activation that can be initiated
directly with phorbol esters or by receptor-stimulated
increases in DAG. Activation of the enzyme might also be
influenced by changes in membrane structure or
constituents[9]. Our results suggest that LPS could activate
DAG-PKC signaling pathway in rat PIM, which is in accordance
with previous reports.
Macrophages stimulated by LPS or other inflammatory
factors produce and release large quantities of various
proinflammatory cytokines, including TNF-a, IL-1b, and
IL-6. Overproduction of the cytokines can result in systemic
inflammatory response syndrome, multiple organ dysfunction
syndrome (MODS) and death. NF-kB plays a pivotal role in
LPS-induced TNF-a and IL-1b gene expressions. The PKC
signaling pathway is linked to the upstream regulation
mechanism of NF-kB activation. In particular, recent findings show
that PKCz can regulate NF-kB through an IkB kinase
(IKK)-independent pathway, by directly phosphorylating
Ser311 of the p65 subunit (RelA), and play an important role in
mediating inflammatory response induced by
LPS[26]. The present study showed that CCK-8 concentration-dependently
inhibited LPS-induced DAG content, PKC activities, and
PKCz translocation, and a significant inhibition was observed at
high concentrations, but not at low concentrations. Cong
et al reported that CCK-8 inhibited LPS-induced
NF-kB binding activity in a concentration-dependent
manner[21]. The present study demonstrated that CCK-8 significantly
inhibited LPS-induced activation of the DAG-PKC signaling
pathway at supraphysiological concentrations, which might be
one of the upstream mechanisms for modulating NF-kB
activity and exerting the anti-inflammatory effect of CCK-8.
However, it is unknown whether the inhibitory effect of
CCK-8 on DAG-PKC signaling pathway induced by LPS was
mediated through the CCK receptor or through which
subtype of its receptor. Cong et al
reported that CCK-8 inhibited NF-kB binding activity and decreased IkB-a
degradation through CCK receptors in rat PIM stimulated by
LPS[21]. In the present study, the inhibitory effect of CCK-8 on
LPS-induced DAG content, PKC activities and PKCz
translocation was abrogated in part by proglumide, CR-1409, and
CR-2945 in a concentration-dependent manner. Proglumide was
so potent that it had an obvious effect on CCK-8-resulted
inhibition of LPS-induced DAG content, PKC activities, and
PKCz translocation at low concentrations, and even caused
a full reversal of it at high concentrations. A similar effect
was found for CR-1409, but not for CR-2945. CR-2945 showed
a weak effect only at high concentrations. Our results
indicate that the inhibitory effect of CCK-8 on the LPS-activated
DAG-PKC signaling pathway in rat PIM was mediated through the CCK receptor, and that both CCK-AR and
CCK-BR might be involved in this pathway. However, CCK-AR
might play a major role in this process, which would be of
benefit in the development of receptor-specific drugs in
clinics.
In conclusion, CCK-8 was a negative modulator of the
DAG-PKC signaling pathway in rat resting PIM, which was
very important for maintaining body homeostasis, and
significantly inhibited LPS-induced DAG content, PKC activity,
and PKCz translocation in a concentration-dependent
manner. CCK receptors, particularly CCK-AR, might
play a major role in this process. Inhibitory effects of CCK-8 on
LPS-induced activation of the DAG-PKC signaling pathway
might be one of the upstream mechanisms for modulating
NF-kB activity and exerting its anti-inflammatory effect in rat
PIM.
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