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INTRODUCTION |
C-type natriuretic peptide
(CNP)1 belongs to the
natriuretic peptide family, which also comprises atrial natriuretic
peptide (ANP) and brain natriuretic peptide. ANP is mainly synthetized in the atrium, whereas brain natriuretic peptide and CNP are more widely produced (1). The three peptides play an important role in the
maintenance of blood pressure and cardiovascular homeostasis and exert
natriuretic and diuretic effects. In addition, natriuretic peptides
exhibit relaxing and growth inhibitory responses in parenchymal and
mesenchymal cells of various origins (for a review, see Ref. 1). In
keeping with the diversity of natriuretic peptide functions, three
classes of receptors have been characterized in different tissues,
natriuretic peptide receptor (NPR)-A, which is sensitive to ANP and
brain natriuretic peptide (2), NPR-B, which is highly specific for CNP
(3), and NPR-C, which binds the three natriuretic peptides with similar
affinities (2). NPR-A and NPR-B are members of the guanylyl cyclase
receptor family and transduce their biological effects via cGMP (2),
whereas NPR-C lacks the guanylate cyclase domain and signals through
inhibition of cAMP (4). Recent data indicate that the mRNAs for
NPR-A, NPR-B, and NPR-C are expressed in human liver (5).
Hepatic stellate cells (HSC) (also known as lipocytes, fat-storing
cells, or perisinusoidal cells) are resident cells in the space of
Disse that show a pericyte-like orientation, extending long cytoplasmic
processes around hepatic sinusoids. Recent studies have advocated their
salient role in the pathogenesis of liver fibrosis and of portal
hypertension (6). Following liver injury, HSC undergo phenotypic
activation from a quiescent cell containing large retinoid droplets to
an activated myofibroblastic-like cell. This state is characterized by
intense proliferation, marked synthesis of extracellular matrix, and
production of proinflammatory cytokines, which lead to the development
of liver fibrosis (6). Proliferation and accumulation of
myofibroblastic HSC (mHSC) have largely been documented in experimental
models and in culture studies (7, 8). Among several mitogenic growth
factors, PDGF-BB, which is highly expressed during chronic hepatic
injury, is currently considered as the most potent mitogen (9, 10).
Factors that may limit the proliferation of myofibroblastic HSC have
also been characterized, such as endothelin-1 (ET-1), cAMP, TNF-
,
and prostaglandins (E2 and I2) (11-13). During phenotypic activation,
HSC also acquire smooth muscle features, such as the expression of
smooth muscle -actin (6), and contract in response to diverse
vasoactive mediators (14-18). Moreover, increasing evidence indicates
that enhanced sensitivity of myofibroblastic HSC to contractile
peptides elevates intrahepatic resistance and contributes to portal
hypertension associated with the development of liver fibrosis
(19).
In the present study, we provide the first evidence for a hepatic
effect of CNP. In a model of human myofibroblastic HSC that displays
the phenotypic characteristics of mHSC found in situ during
hepatic fibrosis (20), we show that activation of NPR-B receptor leads
to elevation of cGMP levels and results in inhibition of mHSC
proliferation and contraction. Analysis of the signaling pathways
indicate that growth inhibition is associated with inhibition of MAP
kinase (extracellular signal-regulated kinase (ERK) and c-Jun
N-terminal kinase (JNK)) activations and blunting of AP-1 binding
stimulation. Relaxing effects of CNP are consecutive to a blockade of
the calcium influx through store-operated calcium channels.
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EXPERIMENTAL PROCEDURES |
Materials
Human CNP-(32-53) was from Bachem (Voísins-le-Bretonneux,
France), and human -ANP-(1-28) was from Neosystem (Strasbourg, France). PDGF-BB was from Life Technologies, Inc. Fetal calf serum was
from Life Technologies, Inc., and pooled human AB positive serum was
supplied by the National Transfusion Center. Hybond N+ membrane, rapid
hybrid buffer, [methyl-3H]thymidine (25 Ci/mmol), and [
-32P]ATP (5,000 Ci/mmol) were from
Amersham Pharmacia Biotech, and 125I-[Tyr0]CNP-22 ([125I]CNP)
(500 Ci/mmol) was from Peninsula (Mersyside, United Kingdom). AP-1 and
NF-kB consensus oligonucleotides, Access RT-PCR kit and Cell Titer 96 Aqueous One Solution cell proliferation assay were from Promega
(Charbonnieres, France). The plasmid encoding glutathione S-transferase-c-Jun (1-79) fusion protein was a generous
gift of Dr. C. Bradham (Chapel Hill, NC). The phospho-p38 MAP kinase antibody was from New England Biolabs (Ozyme, Montigny le Breteonneux, France), cAMP and cGMP radioimmunoassays were from Immunotech (Marseille, France), and Fura-2/AM was from Molecular Probes
(Interchim, Montluçon, France).
8-(4-Chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate,
Rp-isomer (Rp-8-pCPT-cGMP) was from Biolog Life Science Institute
(Bremen, Germany). The protein assay kit was from Bio-Rad. All other
chemicals were from Sigma. HS-142-1 was kindly provided by Dr.
Nakanishi (Kyowa Hakko Kogyo Co., Ltd., Shizuoka, Japan).
Cell Isolation and Culture
Human mHSC in their activated phenotype were obtained by
outgrowth of normal liver explants obtained from surgery of benign or
malignant liver tumors. This procedure was performed in accordance with
ethical regulations imposed by French legislation. Explants were
incubated in Dulbecco's modified Eagle's medium containing 10% serum
(5% fetal calf serum, 5% pooled human serum), and exhaustive characterization of these cells has already been published (20). Cell
isolates were routinely characterized by a positive staining for smooth
muscle -actin, a marker of HSC in their myofibroblastic phenotype.
Experiments were performed between passages 3 and 7, without any
noticeable difference in results observed with cells obtained from
various passages or from various livers. All experiments were performed
on cells made quiescent by a 3-day incubation in serum-free Waymouth medium.
Reverse Transcription and Amplification by PCR
For analysis of NPR, total RNA was extracted in guanidium
isothiocyanate from confluent mHSC made quiescent in serum-free Waymouth medium over 3 days, as described previously (11). Poly(A) RNA
from human kidney and total RNA from human liver and heart were kindly
provided by Drs. F. Bulle and S. Le Gouvello (INSERM U99,
Créteil, France), respectively, and used as positive control. For
each condition, cDNA synthesis and PCR amplification were performed
in the same tube. A cDNA strand was synthetized with reverse
transcriptase using a commercialized kit (Promega), from 5 ng of RNA of
human mHSC and human heart RNAs and 0.5 ng of kidney poly(A) RNA. The
reaction was performed for 45 min at 48 °C, and the cDNA was
used as template DNA for the PCR amplification. In all experiments, the
presence of possible contamination with genomic DNA was tested by
omitting reverse transcriptase from the medium, and the product was
then processed in parallel with the other samples. The oligonucleotide
primers for the human NPR-A (21) (sense,
5'-GGAGCGGACCCAGGCATACCTGGAGG-3'; antisense,
5'-AGGTCAGCCTCGGGTGCTACTC-3') predicted a PCR fragment of 693 bp; the primers for NPR-B (22) (sense, 5'-GGTGGCACCAGCATATTGGACAAC-3';
antisense, 5'-TACAGGAGTCCAGGAGGTCCTT-3') predicted a PCR fragment
of 767 bp; the primers for NPR-C (23) (sense,
5'-GTGGCCCGGCTTGCATCGCACTGGG-3'; antisense, 5'-TCCGGATGGTGTCACTGCTC-3') predicted a PCR fragment of 379 bp. The conditions for PCR
amplification were as follows: denaturation at 94 °C for 1 min,
primer annealing at 55 °C for 1 min, elongation at 72 °C for 1 min. 35 cycles were performed for NPR-A, NPR-B, and NPR-C. PCR products
were size-fractionated in a 2% agarose gel and blotted onto Hybond N+
membrane. After a prehybridization in rapid hybrid buffer (Amersham
Pharmacia Biotech) for 30 min at 42 °C, the membrane was hybridized
in the same buffer for 1 h at 42 °C with 10 ng/ml of
oligonucleotides complementary to sequences within the cDNAs
flanked by the PCR primers, labeled with T4 polynucleotide kinase
(NPR-A probe, 5'-GTACAAGGTGGAGACAATTGGC-3'; NPR-B probe,
5'-CACGCATTGTCAGCAGAGAGCACC-3'; NPR-C probe,
5'-GGGTTTGCACACGTCCATCTA-3'). After washing in 6× SSC, 0.1%
SDS for 15 min at 22 °C and then for 45 min at 42 °C, membranes
were subjected to PhosphorImager analysis (Molecular Dynamics,
Bondoufle, France).
[125I]CNP Binding Assays
Confluent mHSC in six-well plates were made quiescent by a 3 days incubation in serum-free Waymouth medium and further stimulated for 2 h at 4 °C in Waymouth medium containing 0.2% bovine
serum albumin, 1 mM bacitracin, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, with 10 pM of [125I]CNP and varying concentrations of
either unlabeled CNP or ANP. The incubation was stopped by aspirating
the medium and rinsing the cells four times with 4 ml of washing buffer
(20 mM HEPES, pH 7.4, containing 137 mM NaCl
and 1% bovine serum albumin). The cells were then solubilized with 0.4 N NaOH, and cell-associated radioactivity was measured.
Nonspecific binding was determined by incubating with 1 µM CNP, and it usually represented 25% of the total
radioactivity. Experiments were performed in duplicate, and the protein
content was determined by the Bio-Rad protein assay kit in three
separate wells.
cGMP and cAMP Assays
Confluent mHSC were made quiescent by a 3-day incubation in
serum-free Waymouth medium, preincubated with 0.6 mM
isobutylmethylxanthine for 15 min, except otherwise indicated, and
then stimulated for various periods of time in phosphate-buffered
saline containing varying concentrations of natriuretic peptides. cAMP
and cGMP were extracted and assayed as described previously (24), using a commercial radioimmunoassay.
DNA Synthesis and Cell Proliferation Assays
DNA synthesis was measured in triplicate wells by incorporation
of [3H]thymidine, as described previously (11). Confluent
mHSC were made quiescent by a 3 days incubation in serum-free Waymouth
medium and then stimulated for 30 h with the indicated effectors,
in the presence of 80 µM ZnCl2, which has
been described to enhance the growth inhibitory effects of cGMP analogs
(25). [3H]Thymidine (0.5 µCi/well) was added during the
last 6 h of incubation.
Cell growth assay was performed using the Cell Titer 96 Aqueous One
Solution cell proliferation assay (Promega). Human mHSC were seeded in
96-well plates at low density (5000/well) in Dulbecco's modified
Eagle's medium 5/5, allowed to attach overnight, and made quiescent by
a 48 h incubation in serum-free medium. Incubation was performed
in Waymouth medium containing 80 µM ZnCl2 and
either 5% human serum or 20 ng/ml PDGF-BB, in the absence or presence of 100 nM CNP, which was added every day for 3 days. The
medium was then removed for phosphate-buffered saline, Cell Titer 96 Aqueous One Solution reagent was added to each well, and absorbance was
recorded at 490 nm.
Preparation of Whole Cell, Nuclear, and Cytoplasmic Extracts
Whole Cell Extracts--
Whole cell extracts were prepared as
described previously (13) with minor modifications. Confluent mHSC were
made quiescent by a 3-day incubation in serum-free Waymouth medium and
were further incubated for various periods of time with the indicated
effectors. After a wash in ice-cold phosphate-buffered saline, cells
were lysed for 15 min at 4 °C in whole cell extraction buffer (50 mM HEPES, pH 7.4, containing 0.5% Nonidet P-40, 10%
glycerol, 137 mM NaCl, 1 mM EGTA, 10 mM NaF, 1 mM vanadate, 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 40 mM 
-glycerophosphate, 0.1 mM
dithiothreitol). Lysates were centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatants (whole cell
extract) were stored at 
80 °C until use.
Nuclear and Cytoplasmic Extracts--
Nuclear and cytoplasmic
extracts were prepared as described previously (13). Confluent
quiescent mHSC were incubated for various periods of time with the
indicated effectors. Cells were then washed two times in ice-cold
phosphate-buffered saline and resuspended in 400 µl of Buffer A (10 mM HEPES, pH 7.9, containing 1.5 mM
MgCl2,10 mM KCl, 0.5 mM
dithiothreitol, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml
aprotinin, 1 µg/ml pepstatin A). The cells were allowed to swell on
ice for 15 min, after which 12.5 µl of 10% Nonidet P-40 was added.
The tubes were shaken gently, centrifuged at 2000 × g
for 10 min at 4 °C, and supernatants were used as cytoplasmic
extracts. The pellet nuclei were resuspended in 40 µl of Buffer C (20 mM HEPES, pH 7.9, containing 1.5 mM
MgCl2, 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A). After 30 min at 4 °C under constant agitation,
nuclear debris were centrifuged at 20000 × g for 15 min. The supernatants (nuclear extract) were frozen in liquid nitrogen
and stored at 80 °C.
Western Blotting Analysis
Equal amounts of extracts (40 µg) were electrophoresed on a
10% SDS-polyacrylamide gel. Proteins were then electroblotted onto
nitrocellulose membranes and blocked in 10 mM Tris, pH 8, containing 150 mM NaCl, 0.05% Tween 20, 5% skim milk.
Detection of IkB- and phospho-p38 MAP kinase was performed after
incubation for 2 h with their respective antibodies diluted
1:1000. Immunodetected proteins were visualized by using an enhanced
chemiluminescence assay kit (Amersham Pharmacia Biotech) according to
the manufacturer's instructions. Equal loading of proteins in each
lane was checked by Ponceau red staining of the membrane.
Extracellular Signal-regulated Kinase, p38 MAP Kinase, and JNK
Assays
Confluent quiescent mHSC were stimulated with the indicated
effectors, and whole cell lysates were obtained as described above. ERK
activity was assayed in situ, as described previously (11), following electrophoresis of equal amounts of cell lysates (40 µg of
proteins) on a 10% SDS-polyacrylamide gel co-polymerized with 0.5 mg/ml myelin basic protein. JNK was assayed in vitro by the
phosphorylation of glutathione S-transferase-c-Jun (1-79) fusion protein, followed by SDS-polyacrylamide gel, as described previously (12). ERK and JNK activity were quantified by PhosphorImager analysis (Molecular Dynamics). Phosphorylation of p38 MAP kinase was
analyzed by Western blotting, using an antiserum specific to
phospho-p38 MAP kinase.
Electrophoretic Mobility Shift Assay (EMSA)
AP-1 double-stranded consensus oligomer (5'-CGC TTG ATG AGT CAG
CCG GAA-3'; 3'-GCG AAC TAC TCA GTC GGC CTT-5', Promega) and an oligomer
(Promega) corresponding to the consensus sequence of NF-kB from the k
light chain enhancer were radiolabeled with T4 polynucleotide kinase
and [-32P]ATP. Unincorporated nucleotides were removed
by filtration though a G50 Fine column. Nuclear extracts (10 µg of
protein) were incubated in the binding reaction medium (20 mM HEPES, pH 7.9, 100 mM KCl, 20% glycerol,
0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF) for 15 min at 4 °C, followed by a 15-min
incubation at room temperature with 0.5 ng of the
32P-labeled probe. The DNA-protein complexes were analyzed
on a 5% polyacrylamide gel in 0.25 X Tris Borate EDTA electrophoresis buffer. Gels were run at 150 V for 90 min, dried, autoradiographed, and
quantified by PhosphorImager analysis.
Fura-2 Loading and Ca2+ Imaging
Human mHSC were plated at a density of 15,000 cells/ml in 35-mm
dishes, the bottoms of which were replaced by glass coverslips, and
allowed to attach in Dulbecco's modified Eagle's medium 5/5 for
24 h. Cells were made quiescent in serum-free Waymouth medium for
24 h and washed in 121 mM NaCl, 10 mM
HEPES, pH 7.4, 5 mM NaHCO3, 4.7 mM
KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.0 mM CaCl2, 10 mM glucose (Buffer A) containing 1.8% bovine serum
albumin. mHSC were then loaded for 30 min at 25 °C with 1 µM Fura-2/AM in 2 ml of Buffer A containing 1.8% bovine
serum albumin (Buffer B) to improve Fura-2 dispersion and facilitate
cell loading. Unincorporated Fura-2/AM was eliminated by two washes in
Buffer A containing 0.01% bovine serum albumin, and cells were allowed
to incubate in the same buffer for 10 min at 25 °C to facilitate
hydrolysis of intracellular Fura-2/AM. Ca2+ imaging was
performed as described previously (26). Briefly, Fura-2-loaded mHSC
were placed on the stage of a Nikon diaphot inverted microscope with
epifluorescence. Light from a 100-W xenon lamp was filtered alternately
through 360- and 380-nm filters to determine the ratio of fluorescence
F360/F380. Fura-2 fluorescence (Nikon UV-fluor × 40 objective)
was filtered at 510 nm and recorded by an intensified charged-coupled
device Photonic Science camera (27). Each fluorescence image was the
average of two images, to improve the signal-to-noise ratio, and one
average image was recorded every 3 s. All tracings of fluorescence
ratio (F360/F380) are representative of at least 10 cells, and were
performed on at least three different cell preparations. Imaging
studies were performed on cells in which no spontaneous rise in
Ca2+ was observed prior to experimental manipulation. For
experiments performed in absence of extracellular Ca2+, 1 mM EGTA was added to Ca2+-free Buffer B (Buffer C).
Measurement of Cell Contraction
Cell areas and cell lengths were determined from the 360 nm
fluorescence images recorded to measure the F360/F380 ratios of Fura-2-loaded human mHSC, using the Morphostar II software developed by
IMSTAR Co. (Paris, France), as described previously (26). All tracings
of cell areas are representative of at least 10 cells and were
performed on at least three different cell isolations.
Electrophysiology
For patch-clamp experiments, cells were plated on 15-mm
coverslips in 24-well plates at a density of 10,000 cells/well and allowed to attach in Dulbecco's modified Eagle's medium 5/5 for 24 h. Cells were made quiescent in serum-free Waymouth medium for
24 h and the coverslip was mounted on the stage of an inverted microscope. The whole-cell configuration was used to record calcium currents (ICa) with a protocol consisting in a pulse to 0 mV (400-ms duration) preceded by a short pulse of 50 mV (50-ms
duration) elicited every 8 s from a holding potential of 100 mV.
Time-dependent ICa values were measured as
described (28). The cells were voltage-clamped using a patch-clamp
amplifier (Biologic, Grenoble, France), and analyzed as described
previously (28). The experiments were performed at 25 °C.
Solutions
The external solution contained (in mM) 100 NaCl, 10 HEPES, 26 CsCl, 5 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, 2 CaCl2, 10 D-glucose, pH 7.4 adjusted with CsOH.
Solutions were applied as described (28). The patch pipettes (3.5-6.0
Mohm) were filled with an internal solution composed of (in
mM) 130 CsCl, 5 EGTA (acid form), 0.05 CaCl2, 3 Na2ATP, 2 Na2GTP, 10 HEPES, pH 7.2, adjusted
with CsOH, according to Ref. 29.
Assay of Protein Concentration
Protein concentration was determined by the Bio-Rad protein
assay kit.
Statistics
Results are expressed as mean ± S.E. of n
experiments and were analyzed by repeated measures ANOVA.
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RESULTS |
Characterization of Natriuretic Peptide Receptor Subtypes in Human
Myofibroblastic HSC--
Identification of the natriuretic peptide
receptor subtypes present in human myofibroblastic HSC was performed by
RT-PCR analysis. The mRNA was amplified with specific primers
complementary either to the human NPR-A or to the NPR-B or NPR-C DNA
sequences, and the PCR product was size-fractionated and blotted. The
membrane was hybridized with a labeled oligonucleotide complementary to the respective NPR sequences within the cDNA flanked by the PCR primers. A band of 767 bp corresponding to the size of the NPR-B product was identified in human myofibroblastic HSC (Fig.
1A). In contrast, mHSC did not
expressed the expected 693-bp product or a 379-bp product corresponding
to NPR-A or NPR-C, whereas these amplification products were present in
human kidney taken as control (Fig. 1A). The functionality
of NPR-B in myofibroblastic HSC was assessed in binding experiments.
Competition experiments indicated that CNP was more potent than ANP in
inhibiting [125I]CNP binding, with IC50
values of 20 and 800 pM, respectively (Fig. 1B).
This order of potency and these IC50 values are in agreement with those described for NPR-B (3).
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Fig. 1.
Characterization of the presence of NPR in
human myofibroblastic HSC. A, detection of NPR
mRNAs by RT-PCR. The products of PCR amplification of cDNAs
from human myofibroblastic HSC, human heart, and human kidney were
prepared as described under "Experimental Procedures,"
electrophoresed on a 2% agarose gel, and blotted. The membrane was
hybridized with a labeled oligonucleotide complementary to the
respective NPR sequences within the cDNA flanked by the PCR
primers, as described under "Experimental Procedures." The
arrows point to 693-, 767-, and 379-bp fragments
corresponding to NPR-A, NPR-B, and NPR-C cDNAs, respectively.
B, characterization of NPR-B by competition experiments.
Inhibition of [125I]CNP binding by unlabeled CNP or ANP
was performed on confluent mHSC made quiescent by a 2-day incubation in
serum-free Waymouth medium, as described under "Experimental
Procedures." Results are the mean of two experiments. C,
effect of CNP and ANP on cGMP levels. Confluent mHSC were made
quiescent in serum-free Waymouth medium over 3 days and were further
incubated for 10 min (i) with varying concentrations of either CNP or
ANP (top panel) or (ii) with or without 20 ng/ml PDGF-BB in
the absence or presence of 100 nM CNP (bottom
panel). cGMP was assayed as described under "Experimental
Procedures." Results are the mean ± S.E. of three experiments.
For ANP (B), error bars are included in the
symbol. p < 0.01 by repeated measures ANOVA.
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In keeping with the presence of NPR-B receptors, and as described in
other cells (3), CNP caused a huge (30-fold) increase in cGMP levels in
mHSC, whereas ANP elicited a minimal (1.6-fold) effect (Fig.
1C). cGMP levels were also measured in the conditions used
in DNA synthesis assays, i.e. in the presence of PDGF-BB (see below). Whereas PDGF-BB alone had no effect, CNP increased cGMP
levels to the same extent in the presence of PDGF-BB and in its absence
(Fig. 1C).
We also determined whether elevation of cGMP increases cAMP levels, via
activation of the cGMP-inhibited phosphodiesterase. Blockade of the
cGMP-inhibited phosphodiesterase by 10 µM milrinone (30)
did not affect cAMP levels, indicating a negligible participation of
the cGMP-inhibited phosphodiesterase in cAMP metabolism in human mHSC.
Moreover, when phosphodiesterases were blocked by the nonselective
inhibitor isobutylmethylxanthine, neither CNP nor 8-Br-cGMP affected
cAMP levels, even after a prolonged 90-min stimulation (Table
I); in contrast, as expected (12), the
endothelin B receptor agonist sarafotoxin S6C caused a 6-fold increase
in cAMP levels. These results indicate that cGMP mobilizing agonists do
not signal through cAMP in human myofibroblastic HSC.
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Table I
cGMP mobilizing agonists do not affect cAMP levels in human
myofibroblastic HSC
Confluent mHSC were made quiescent in serum-free Waymouth medium over 3 days, and were further preincubated with 0.6 mM
isobutylmethylxanthine for 15 min, except for milrinone experiments, in
which isobutylmethylxanthine was omitted. Cells were then stimulated
for 10 min with the indicated agonists, unless otherwise indicated.
cAMP levels were assayed as described under "Experimental
Procedures." Results are the mean ± S.E. of three experiments.
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Altogether, these data demonstrate the presence of functional NPR-B
receptors in human myofibroblastic HSC. The next series of experiments
were performed to investigate the biological functions of NPR-B
receptors in human myofibroblastic HSC and focused on the effects of
CNP on their proliferation and contraction.
CNP Inhibits the Growth of Human Myofibroblastic HSC via NPR-B
Receptor--
DNA synthesis of human myofibroblastic HSC was
stimulated with the most potent mitogens for human mHSC, either human
serum (5%) or the purified growth factor PDGF-BB (20 ng/ml). (Fig.
2A). DNA synthesis of serum-
or PDGF-BB-stimulated cells was reduced dose-dependently by
CNP, a maximal 30% inhibition being attained at 10 nM,
with an IC50 of 30 pM, in agreement with the
IC50 of CNP for NPR-B. In contrast, 10 nM ANP
minimally inhibited [3H]thymidine incorporation. Similar
results were obtained in cell proliferation assays, indicating that
inhibition of [3H]thymidine incorporation is associated
with inhibition of cell growth (Fig. 2B). Addition of
permeant analogs of cGMP reproduced the growth inhibitory effect of
CNP, with cGMP 8-Br-cGMP or 8-CPT-cGMP inhibiting thymidine
incorporation of serum-stimulated mHSC by 35 and 30%, respectively
(Fig. 2C). Finally, the growth inhibitory effect of CNP was
markedly reduced by Rp-8-pCPT-cGMP, a protein kinase G inhibitor, as
well as by HS-142-1, a guanylyl cyclase-coupled receptor antagonist
(31) (Fig. 2D). Taken together, these data indicate that CNP
inhibits the proliferation of human myofibroblastic HSC, following
binding of the peptide to the guanylyl cyclase receptor NPR-B and the
resulting elevation of cGMP.
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Fig. 2.
CNP inhibits DNA synthesis and cell growth of
human myofibroblastic HSC. A, effect of CNP and ANP on
[3H]thymidine incorporation into DNA. Confluent mHSC were
made quiescent in serum-free Waymouth medium over 3 days and were
further stimulated for 30 h in the presence of 80 µM
Zn Cl2 with 20 ng/ml PDGF or 5% human serum
(inset), in the absence or presence of varying
concentrations of CNP or ANP. Results are the mean ± S.E. of nine
experiments. p < 0.01 for CNP and ANP effects by
repeated measures ANOVA. B, effect of CNP on mHSC growth.
Human mHSC were seeded in 96-well plates at low density (5000/well) in
Dulbecco's modified Eagle's medium 5/5, allowed to attach overnight,
and made quiescent by a 48-h incubation in serum-free medium.
Incubation was performed in medium containing 80 µM Zn
Cl2 and either 20 ng/ml PDGF-BB or 5% human serum, in the
absence or presence of 100 nM CNP, which was added every
day for 3 days. Cell growth was assayed at day 3. Results are the
mean ± S.E. of six experiments. p < 0.01 compared with the respective controls by two-way ANOVA for repeated
measures. C, effect of the cGMP analogs 8-CPT-cGMP and
8-Br-cGMP on [3H]thymidine incorporation into DNA.
Quiescent cells were stimulated over 30 h in medium containing 80 µM Zn Cl2 and 5% human serum in the absence
or presence of 1 mM 8-CPT-cGMP or 5 mM
8-Br-cGMP. Results are the mean ± S.E. of six experiments.
p < 0.01 by two-way ANOVA for repeated measures.
D, effect of the protein kinase G inhibitor
Rp-8-pCPT-cGMP and of the NPR-A/B antagonist HS-142-1 on the growth
inhibitory effect of CNP. Quiescent cells were preincubated for 30 min
either with medium alone or with medium containing 100 µM
Rp-8-pCPT-cGMP or 100 µg/ml HS-142-1, and further stimulated over
30 h in medium containing 80 µM Zn Cl2,
20 ng/ml PDGF-BB, and varying concentrations of CNP. Results are the
mean ± S.E. of four experiments. **, p < 0.01 by
two-way ANOVA for repeated measures.
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CNP Inhibits Both ERK and JNK and Has No Effect on p38
MAPK--
We have previously shown that in human myofibroblastic HSC,
antiproliferative effects of ET-1 are associated with inhibition of two
enzymes of the MAPK cascade, ERK and JNK (11, 12). Moreover, another
enzyme of the MAPK family, p38 MAPK, has recently been associated with
growth arrest (32). We therefore investigated the effects of CNP and
8-CPT-cGMP on the activation of ERK, JNK, and p38 MAP kinase. CNP alone
had no effect on either ERK, JNK, or p38 MAPK. PDGF-BB rapidly
stimulated ERK, inducing a maximal activation within 10-15 min
followed by a decrease thereafter (Fig.
3A). In the presence of CNP,
activation of ERK by PDGF-BB was reduced by 30-40% at all time
points. JNK was time-dependently activated by PDGF-BB, a
maximal 3-fold increase being attained after 10-20 min (Fig.
3B). Addition of CNP to PDGF-BB-stimulated cells reduced JNK
activity by 40% (Fig. 3B). CNP also caused a 50% reduction
in JNK activity stimulated by human serum (not shown). Finally, whereas
serum increased the phosphorylation of p38 MAPK, there was no effect of
CNP on serum-stimulated p38-MAPK phosphorylation (Fig. 3C).
ANP had no effect on ERK and JNK in cells stimulated by PDGF-BB (not
shown). As shown in Fig. 4, the cGMP
analog 8-CPT-cGMP (1 mM) reproduced the inhibitory effects
of CNP on ERK and JNK activation by PDGF-BB.
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Fig. 3.
Effect of CNP on ERK, JNK, and p38 MAPK
activations in human myofibroblastic HSC. Confluent mHSC were made
quiescent in serum-free Waymouth medium over 3 days and were further
stimulated with 20 ng/ml PDGF-BB or 5% human serum as indicated, in
the absence or the presence of 1 µM CNP. In C,
cells were stimulated for 15 min with the indicated factors. Cell
lysates were prepared as described under "Experimental Procedures"
and assayed for ERK activity by in gel kinase assay, carried out as
described under "Experimental Procedures" and quantified by
PhosphorImager analysis (A). B, JNK activity,
carried out with glutathione S-transferase-c-Jun as
substrate as described under "Experimental Procedures" and
quantified by PhosphorImager analysis. C, P38 MAPK, analyzed
by Western blotting, using an antiserum specific to phospho-p38 MAP
kinase. Autoradiograms are representative of two experiments and were
quantified by PhosphorImager analysis.
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Fig. 4.
Effect of 8-CPT-cGMP on the activation of ERK
and JNK in human myofibroblastic HSC. Confluent mHSC were made
quiescent in serum-free Waymouth medium over 3 days and were further
stimulated with 20 ng/ml PDGF-BB in the absence or the presence of 1 mM 8-CPT-cGMP for 15 min. Cell lysates were prepared as
described under "Experimental Procedures" and assayed for ERK
activity (A) and JNK activity (B) as described in
the legend to Fig. 3. The autoradiograms shown are representative of
two experiments and were quantified by PhosphorImager analysis.
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CNP Inhibits AP-1 DNA Binding and Has No Effect on IKB
Degradation--
We have recently described that the antiproliferative
effects of ET-1 and TNF- involves activation of NF-
B and
reduction of AP-1 proteins activation in human myofibroblastic HSC (12, 13). We therefore investigated the effects of CNP on both transcription factors. Nuclear proteins were isolated from human myofibroblastic HSC
treated either with CNP alone, PDGF-BB, or PDGF-BB together with CNP,
and analyzed in EMSAs, using a radiolabeled DNA probe containing a
consensus AP-1 binding sequence. As shown in Fig. 5A, PDGF-BB increased AP-1 DNA
binding time-dependently, with a peak at 30-60 min. CNP
markedly decreased AP-1 DNA binding stimulated by PDGF-BB, whereas it
did not affect basal AP-1 binding. Similar results were obtained when
8-Br-cGMP was used instead of CNP (not shown). The increase in AP-1
binding and in DNA synthesis by PDGF-BB was blocked by PD98059, an
inhibitor of the ERK pathway, thus demonstrating the role of ERK in the
mitogenic pathway stimulated by PDGF-BB (Fig. 5B).
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Fig. 5.
Effects of CNP on AP-1 activation and
IkB- expression in human myofibroblastic
HSC. A, EMSA of AP-1 in nuclear extracts of human mHSC
treated with CNP and PDGF-BB. Nuclear extracts were prepared from mHSC
made quiescent by incubation in serum-free medium over 3 days and
further incubated for various periods of time with either 1 µM CNP, 20 ng/ml PDGF-BB, or PDGF together with CNP. EMSA
was performed as described under "Experimental Procedures," using a
radiolabeled probe containing the AP-1 motif. The autoradiogram shown
is representative of two experiments and was quantified by
PhosphorImager analysis. B, effect of the MEK inhibitor
PD98059 on the stimulation by PDGF-BB of AP-1 DNA binding and DNA
synthesis. Left, nuclear extracts were prepared as in
A from cells pretreated for 30 min with or without 20 µM PD98059 and further incubated for 60 min with 20 ng/ml
PDGF-BB. EMSA was performed as in A. The autoradiogram shown
is representative of two experiments. Right, confluent mHSC
made quiescent in serum-free Waymouth medium over 3 days were
pretreated for 30 min with or without 20 µM PD98059 and
were further stimulated for 30 h in the presence of 20 ng/ml PDGF.
Results are the mean ± S.E. of three experiments. C,
effect of CNP on IkB- expression. Cytoplasmic extracts were obtained
from confluent quiescent mHSC incubated for various periods of time
with 1 µM CNP or 50 ng/ml TNF-. Western blot
measurements of IkB- in the cytoplasmic extracts were performed as
described under "Experimental Procedures," using a specific IkB-
antibody. D, effect of CNP on NF-kB DNA binding. Nuclear
extracts were prepared as in A from cells treated for
various periods of time with 1 µM CNP or 50 ng/ml
TNF-. EMSA was performed as described under "Experimental
Procedures," using a radiolabeled probe containing the NF-kB
motif.
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We also examined the effects of CNP on NF-kB by studying the
degradation of its inhibitory protein IB-, an event that reflects NF-B activation (33), and on NF-B DNA-binding (Fig. 5,
C and D). In unstimulated cells, a 37-kDa
IB- protein was detected in cytoplasmic extracts (Fig.
5C). CNP did not affect IB- levels, whereas TNF-
caused degradation of IB- after 30 min, as expected (Fig.
5C). Moreover, CNP did not affect the DNA binding activity of NF-B, whereas TNF- had a strong stimulatory effect (Fig. 5D).
Altogether, these results indicate that the growth inhibitory effects
of CNP and cGMP are associated with a reduction of ERK and JNK
activation and the blockade of the resulting elevation of AP-1 DNA binding.
CNP Inhibits Thrombin-induced Contraction of Human Myofibroblastic
HSC by Blocking Thrombin Stimulation of Calcium Influx through
Store-operated Calcium Channels--
We investigated the effects of
CNP on the contraction of human myofibroblastic HSC in response to
thrombin, one of the most potent contractile agonists for these cells
(15). As expected, addition of 1 units/ml thrombin caused a transient
contraction of human mHSC, indicated by marked reduction in cell area
(14.7 ± 1.5%, n = 10, Fig.
6A) and cell length (11.5 ± 1.3%, n = 10) (not shown), in 100% of cells.
Preincubation of mHSC for 10 min with 1 µM CNP totally
blunted thrombin-induced mHSC contraction in 100% of cells (Fig.
6B).
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Fig. 6.
Effects of CNP, 8CPT-cGMP, and blockade of
calcium influx on cell contraction and [Ca2+]i in
individual Fura-2-loaded human myofibroblastic HSC. Human
myofibroblastic HSC were made quiescent by incubation in serum-free
medium over 1 day and loaded with Fura-2 as described under
"Experimental Procedures." Fura-2-loaded cells were preincubated
for 10 min with either control buffer (A), 1 µM CNP (B), 1 mM 8-CPT-cGMP
(C), or 1 mM EGTA (D) and further
incubated with 1 unit/ml thrombin. In the upper part of each
panel, cell areas were determined from the 360 nm fluorescence images
recorded to measure the F360/F380 ratios of Fura-2-loaded human mHSC,
using the Morphostar II software as described under "Experimental
Procedures." Ca2+ imaging (F360/F380 ratio) is shown in
the lower part of each panel and was performed on
Fura-2-loaded cells, as described under "Experimental Procedures."
For comparison, the effects of thrombin alone are shown as a
dashed line. All tracings of cell areas and F360/F380 ratios
are representative of at least 10 cells and were performed on at least
three different cell isolations.
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Because contraction of human mHSC is associated with elevation of
intracellular calcium (15), we investigated the effects of CNP on
thrombin-induced increase in [Ca]i. As shown in Fig.
6A, thrombin caused an initial rapid elevation in
[Ca]i, which was followed by a more sustained phase.
Incubation of mHSC with CNP shortened the duration of the calcium peak
induced by thrombin (Fig. 6B). In thrombin-stimulated cells,
the calcium peak lasted 107 ± 7 s and decreased to 76 ± 4 s in thrombin-stimulated cells treated with CNP. In contrast,
CNP modified neither the first phase of the calcium response nor the
amplitude of the calcium transient induced by thrombin. The effects of
CNP were reproduced by addition of the permeant analog of cGMP,
8-CPT-cGMP, which abolished the contractile effect of thrombin and
shortened the duration of the calcium peak (Fig. 6C). Like
CNP, the cGMP analogs did not affect the first phase of the calcium
response, while diminishing the duration of the calcium peak induced by
thrombin to 76 ± 8 s (Fig. 6C).
In nonexcitable cells, the initial phase of the calcium response is due
to release of calcium from intracellular stores, whereas the second
phase of the calcium transient is consecutive to influx of calcium from
external medium (34). In order to investigate the importance of calcium
influx in mHSC contraction, we examined the response of mHSC to
thrombin in the absence of external calcium, i.e. in a
calcium-free medium containing 1 mM EGTA. Treatment with
EGTA shortened the calcium peak elicited by thrombin to 72 ± 6 s, without affecting either the amplitude of the calcium
transient or the first phase of the calcium response (Fig.
6D). Concomitantly, preincubation of mHSC in the
calcium-free medium blunted the contractile effects of thrombin (Fig.
6D), thereby reproducing the effects of CNP.
The next series of experiments were designed to characterize the nature
of the calcium channel responsible for calcium influx and inhibited by
CNP. Voltage-activated calcium currents have been occasionally observed
in hepatic stellate cells from rat origin (29, 35). Therefore, we used
the patch-clamp technique to examine the presence of low voltage
(T-type) and high voltage (L-type) activated calcium channels in human
myofibroblastic HSC, with a two-step protocol. High voltage activated
calcium currents were present, but only in one-third of the cells
studied (Table II), whereas 99% of the
cells were positive for smooth-muscle actin (20); low voltage
activated calcium currents were not detectable. Moreover, thrombin (1 unit/ml) did not stimulate either high voltage or low voltage activated
currents (not shown). Thus, the activity of voltage-gated calcium
channels cannot provide a mechanism for the transmembrane calcium
influx described above. We therefore examined the potential
contribution of store-operated calcium channels in calcium influx
stimulated by thrombin and used a calcium-free/calcium readdition
protocol, which is a sensitive procedure to measure changes in calcium
influx through these channels (36, 37). Fura-2-loaded cells were
stimulated by thrombin in the absence of extracellular calcium. Once
the rapid and transient elevation in [Ca]i had returned to
basal levels, readdition of calcium was performed and resulted in a
fast [Ca]i rise, indicating that intracellular depletion by
thrombin triggers a secondary calcium influx through store-operated
calcium channels (Fig. 7A, 8 out of 11 cells). This influx was totally blocked by addition of 10 nM CNP (Fig. 7B, 12 cells over 14). In addition, it was also totally abolished by LaCl3, a store-operated
calcium influx blocker (37, 38) (Fig. 7C, 8 cells over 11).
In contrast, nitrendipine, a voltage-operated calcium channel
inhibitor, had no effect (Fig. 7D, 14 cells over 18). Taken
together, these results indicate the essential role of calcium influx
in the contractile process and suggest that CNP and 8-CPT-cGMP prevent
mHSC contraction by blocking the influx of calcium through
store-operated calcium channels.
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Table II
Calcium currents in human hepatic stellate cells
The whole-cell patch-clamp technique was performed in the conditions
described under "Experimental Procedures."
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Fig. 7.
CNP inhibits calcium influx through
store-operated calcium channels in human myofibroblastic HSC.
Human myofibroblastic HSC were made quiescent by incubation in
serum-free medium over 3 days and loaded with Fura-2 as described under
"Experimental Procedures." A, calcium store depletion by
thrombin triggers calcium influx through store-operated calcium
channels: Fura-2-loaded cells were preincubated for 10 min with 1 mM EGTA and further incubated with 1 unit/ml thrombin. Once
the rapid and transient elevation in the F360/F380 ratio had fallen
down to basal levels, cells were washed, and 10 mM
Ca2+ was reintroduced in the medium. B, CNP
inhibits store-operated calcium influx. The protocol was as in
A, except that 10 nM CNP was added together with
EGTA and remained present throughout the experiment. C,
LaCl3 inhibits store-operated calcium influx. The protocol
was as in A, except that 0.5 mM
LaCl3 was introduced together with Ca2+.
D, nitrendipine does not affect store-operated calcium
influx. The protocol was as in A, except that 1 µM nitrendipine was added together with EGTA and remained
present throughout the experiment .
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DISCUSSION |
We show here that human myofibroblastic HSC express C-type NPR-B.
Activation of this receptor leads to inhibition of both growth and
contraction of mHSC.
In human liver, mRNAs for the three types of natriuretic peptide
receptors, NPR-A, NPR-B, and NPR-C, have been identified by RT-PCR (5).
Whereas no information concerning the hepatic cell expressing NPR-B has
been provided, biologically active NPR-A and NPR-C have been detected
in liver. Thus, binding of ANP to NPR-C inhibits proliferation of
hepatoblastoma Hep G2 cells (39). Also, NPR-A and NPR-C are coexpressed
in human biliary cells, and NPR-A is coupled to Cl
channels (40). Finally, both NPR-A and NPR-C binding sites are
increased during liver regeneration (41). We show that human myofibroblastic HSC exclusively express the NPR-B mRNA, as
demonstrated by RT-PCR detection. This receptor is functional, as
indicated both in binding experiments and by the dramatic increase in
cGMP levels elicited by CNP as compared with the modest effect of ANP, a typical order of potency of natriuretic peptides for NPR-B.
Growth inhibitory properties of natriuretic peptides have been
described in various cells, but the receptor involved is clearly cell-specific. Thus, the antiproliferative effects of natriuretic peptides are mediated by NPR-B in chondrocytes and NIH3T3 fibroblasts (42, 43), NPR-A in cardiac fibroblasts (44), and NPR-C in mesangial,
vascular smooth muscle, osteoblastic, and astroglial cells (45-48).
The molecular mechanisms involved include inhibition of ERK by NPR-C
and NPR-A (49) and inhibition of JNK and of AP-1 binding via NPR-A, as
recently described in mesangial cells (50). In human myofibroblastic
HSC, CNP is far more potent than ANP in inhibiting mHSC proliferation,
and the IC50 of CNP for inhibiting mHSC growth is similar
to that of CNP for its receptors. The antiproliferative effects of CNP
are reproduced by cGMP analogs and blocked by the protein kinase G
inhibitor Rp-8-pCPT-cGMP, as well as by HS-142-1, an antagonist of
guanylyl cyclase-coupled receptor (31). Growth inhibitory effects of
CNP are associated with inhibition of ERK and JNK and blockade of DNA
binding activity of AP-1; these effects are reproduced by permeant
analogs of cGMP. This suggests that inhibition of ERK and JNK by CNP
are crucial events in the blockade of AP-1-activated genes. The
mechanism by which CNP inhibits ERK and JNK in human mHSC is unknown
but may involve induction of the MAPK phosphatase MKP-1, as reported for ANP in mesangial cells (51), and/or inhibition of receptor tyrosine
kinase by CNP, as observed for the PDGF receptor in smooth muscle cells
(52).
In human myofibroblastic HSC, a part of the signaling pathways
stimulated by cGMP-elevating agonists is common to those stimulated by
cAMP-dependent agonists. Thus, CNP, which increases cGMP,
and ET-1, which activates a prostaglandin/cAMP pathway (12), both cause mHSC growth arrest by blocking ERK and JNK activations and the
resulting AP-1 DNA binding activity. In contrast, whereas ET-1
stimulates NF-B and activates cyclooxygenase-2 (13), CNP has no
effect on either NF-B (Fig. 5) or cyclooxygenase-2 (not shown).
However, the question arises of whether CNP may increase cAMP via cGMP.
This is unlikely, based on two lines of evidence: (i) CNP and 8-Br-cGMP
do not increase cAMP, even after prolonged incubation; (ii) the
activity of cGMP-inhibited phosphodiesterase, which is inhibited by
cGMP and hydrolyzes cAMP (30) is negligible in human myofibroblastic
HSC (Table I). These results demonstrate that blockade of both the
ERK/JNK cascade and AP-1 DNA binding are common crucial steps in the
growth inhibitory effects of both cGMP-elevating factors and
cAMP-mobilizing agonists.
Contraction of myofibroblastic HSC in culture has been reported in
response to ET-1 via endothelin-A receptors, thrombin, angiotensin II,
substance P, thromboxane A2, and vasopressin (14-17), and
contractility is counteracted by relaxing agents, such as c-GMP
elevating factors (nitric oxide or interleukin-1 (53, 54)), and
cAMP-elevating agonists (PGE2, PGI2, and adrenomedullin (16, 55)).
Although vasodilating properties of natriuretic peptides have been
found in diverse tissues (1), few studies have investigated their
effects on hepatic hemodynamics. In normal liver, ANP regulates
intrahepatic resistance because it antagonizes the increase in portal
pressure elicited by 1-adrenergic (56). In rats with
experimental cirrhosis, systemic administration of CNP reduces portal
pressure (57), but whether this effect relates to a decrease in
intrahepatic resistance has not been determined. Our results indicate
that CNP blunts the constrictive effect of thrombin in cultured human
myofibroblastic HSC by reducing both cell length and area, via an
increase in cGMP. This relaxing effect of CNP is associated with a
reduction of the duration of the calcium peak elicited by thrombin, an
effect that is reproduced by chelating calcium from the extracellular
medium. Inhibition of calcium influx by natriuretic peptides has been
reported. However, few studies have characterized the nature of the
calcium channel involved, and either inhibition of L-type
calcium channels in myocytes or T-type calcium currents in glomerulosa
cells have been reported (58, 59). In the present study, modulation of
voltage-operated calcium channels cannot provide a mechanism for the
calcium influx stimulated by thrombin and blocked by CNP because (i)
thrombin stimulation of calcium influx was insensitive to the
L-type calcium channel blocker nitrendipine; (ii) high
voltage gated calcium channels (L-type) recorded with the
patch clamp technique were only occasionally detected, as described by
others in rat HSC (29, 35); and (iii) low voltage activated calcium
channels (T-type) were undetectable. Therefore, we hypothesized that
store-operated calcium channels may be the CNP target and used a
calcium-free/calcium readdition protocol. This protocol has been used
in several cell types (see, for example, Refs. 36 and 37) and relies on
the fact that either calcium mobilization or depletion of calcium from
endogenous stores constitutes an essential step for stimulation of
store-operated calcium channels (SOC). We observed that thrombin stimulation of SOC in human mHSC was totally blocked by CNP as well as
by a SOC inhibitor, LaCl3. Taken together, these data strongly suggest that CNP relaxes myofibroblastic HSC following blockade of calcium influx through store-operated calcium channels. These results, which constitute the first report of store-operated calcium channels as a target for CNP, provide a new mechanism for the
relaxing effects of CNP.
Recent evidence supports a role for myofibroblastic HSC in the
regulation of sinusoidal tone, and thereby of intrahepatic resistance
(19). Modulation of sinusoidal tone results from the fine tuning
between contractile and relaxing activities of diverse mediators
produced locally. Little is known regarding hepatic production of CNP.
CNP mRNA is expressed in human liver (5), suggesting the existence
of a local natriuretic peptide system, but cellular origin of the
peptides remains to be determined. Our preliminary data suggest that
hepatocytes are a major source of CNP in human liver and that its
expression is increased during chronic liver
diseases.2 These results
suggest that during chronic liver injury, CNP could play a key role in
counteracting liver fibrosis and associated portal hypertension by
inhibiting mHSC proliferation and antagonizing the contractile response
of these cells to vasoactive mediators.
,
TOP
ABSTRACT
INTRODUCTION
icale and the Ligue
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