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Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Königinstrasse 16,D-80539 Munich, Germany
Address all correspondence and requests for reprints to: Angelika M. Vollmar, Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Königinstrasse 16, D-80539 Munich, Germany. E-mail: vollmar{at}pharmtox.vetmed.uni-muenchen.de
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This information on differential regulatory mechanisms controlling the NP and their receptors supports the idea of distinct physiological functions for the NP.
Our previous work drew attention to a new aspect in the biological profile of the NP family: the interference with the immune system. ANP but not CNP was demonstrated to inhibit thymocyte proliferation (18) and thymopoesis (19). Furthermore, ANP stimulates phagocytosis and production of reactive oxygen by macrophages (20). In addition, we could recently show that ANP is able to inhibit the production of nitric oxide (NO) in primary mouse macrophages activated by lipopolysaccharide (LPS) (21).
The aim of the present study was 1) to compare the data on the NO-inhibitory effect of ANP in primary macrophages with those in the mouse macrophage cell lines RAW 264.7 and J774. 2) The question whether inhibition of NO synthesis by macrophages is specific for the atrial natriuretic peptide or shared by other members of the NP family should be examined. Thus, an ANP fragment, atriopeptin I (API, ANP 102–123) (22), urodilatin as well as CNP were investigated for their NO-inhibitory effect. 3) The study aimed to determine the receptor specificity of the NO-inhibition by ANP. In this regard, mRNA expression of all three NPR was demonstrated for the first time in macrophages. Next, the effect of des-(Gln18, Ser19, Gly20, Leu21, Gly22)-ANF 4–23 (C-ANF), a C-type receptor ligand (5) and of HS-142–1, a NPR-A/B receptor antagonist (23) on NO-production has been evaluated. Additionally, stable analogs of the second messenger cGMP (8-Br-cGMP, dibutyryl-cGMP), as well as LY-83583, an antagonist to cGMP generation (24) have been employed. 4) Finally, information on the time dependency of the NO-inhibition by ANP was obtained.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Cell lines.
The murine macrophage cell lines RAW 264.7
(American Type Culture Collection ATCC, TIB 71, Rockville, MD) and J
774 (ATCC, TIB 67) were cultured in DMEM (Bio Whittaker, Bioproducts,
Heidelberg, Germany) containing 4 mM glutamine and 10%
heat-inactivated FCS (GIBCO-BRL, Eggenstein, Germany). DMEM used for
the J 774 cells did not contain phenol red. Cells were grown at 37 C
and with 5% CO2 in fully humidified air and used for
experiments between passage 5 and 20.
Primary cell cultures.
Bone marrow macrophages (BMM) were
prepared as described previously (25) and cultured in RPMI-1640 medium
supplemented with 20% (vol/vol) L-929 cell conditioned medium (LCM) as
a source of the macrophage growth factor, 10% heat-inactivated FCS,
and penicillin (100 U/ml)/streptomycin (100 µg/ml). 2 x
105 cells were seeded in 24-well tissue plates (Peske,
Aindling-Pichl, Germany) and grown for 5 d (5% CO2,
37 C). Cells were made quiescent by removing LCM at least 12 h
before the experiment.
Peritoneal macrophages (PM) were collected by abdominal lavage (RPMI 1640 containing 10% FCS) of mice treated with 4% thioglycollate broth (ip, 1 ml) for 4 days (25). Cells were seeded in 24-well tissue plates (1 x 106 cells/well). After 2 h incubation (37 C, 5% CO2), nonadherent cells were removed and fresh medium added.
BMM and PM preparations were found >95% pure as judged by FACS analysis (Becton Dickenson, San Jose, CA), using an antiserum against the macrophage antigen F40/80 (Serotec LTD, Wiesbaden, Germany) (26).
Measurement of nitrite accumulation (Griess assay)
BMM and PM were grown in 24-well plates, RAW 264.7 and J 774
cells in 96-well plates (Peske). Confluent cells were treated with
bacterial lipopolysaccharide (LPS, E. coli, serotype 055:B5,
1 µg/ml) in the presence or absence of various concentrations of NP
or other indicated substances. Test substances were dissolved in medium
and stored (-70 C) in BSA-coated tubes, except for LY 83583, which was
dissolved in DMSO. Final DMSO concentration on the cells was <0.01%
and was shown not to interfere with the assay. After 20 h, the
concentration of nitrite, a stable metabolite of NO, was measured in
the culture supernatant by the Griess reaction (27) as follows: 100
µl of supernatant was removed and 90 µl 1% sulfanilamide in 5%
H3PO4 and 90 µl 0.1%
N-(1-naphthyl)ethylenediamine dihydrochloride in
H2O was added, followed by spectrophotometric measurement
at 550 nm (reference wavelength 620 nm) using a SPECTRA microplate
reader (SLT-Labinstruments, Heidelberg, Germany). Nitrite
concentrations were determined by comparison with a standard curve of
sodium nitrite in medium.
Cytotoxicity assay
Mitochondrial reduction of 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to
formazan was determined as an indicator of cell viability (28). After
removing the supernatant for nitrite determination, the cells were
incubated with MTT (0.5 mg/ml) for 1 h at 37 C and solubilized in
DMSO. The extent of formazan production was determined photometrically
at 550 nm.
Degradation of NP by macrophages
Stability of the peptides during incubation with macrophages was
determined by incubating BMM, RAW 264.7 and J774, respectively with
approximately 30000 cpm125I-labeled ANP or
125I-CNP in the presence of 10 nM of the
corresponding unlabeled peptides. Untreated as well as LPS-stimulated
(1 µg/ml) macrophages were employed. Furthermore, degradation of NP
was determined in LPS-activated cells pretreated (1 h) with thiorphan
(10-5 M) or phosphoramidon (10-5
M), or the combination of both. Supernatants (n = 3)
were collected at various times after addition of NP and analyzed by
reverse phase-HPLC (C18 Bondapak column; 4.5 x 2500
mm). Elution of ANP and CNP material was conducted with a linear
gradient of 20–55% acetonitrile in 0.1% TFA (55 min, flow rate 1
ml/min). Degradation of peptides during culture was estimated by
counting radioactivity of HPLC fractions and comparing the profile and
amount of eluted radioactivity with that of intact radiolabeled
peptide.
Analysis of NPR-mRNA in macrophages
mRNA extraction and cDNA synthesis.
Total RNA of macrophages
was isolated by the guanidinium thiocyanate/cesium chloride method and
mRNA was purified by means of poly-dT adsorption (PolyATract kit,
Promega, Heidelberg, Germany) as previously described in detail (9, 25). mRNA (1 µg) was transcribed to cDNA using avian myeloblastosis
virus reverse transcriptase (Promega) according to established
protocols (9, 25).
Oligonucleotides used for PCR amplification.
All
oligonucleotides were obtained from MWG (Ebersberg, Germany) and had
been HPLC-purified. For the NPR-A mRNA amplification, the following
oligonucleotides were used based upon the sequence for the mouse NPR-A
gene (29); upstream: 5'-AAGAACCCGATAATCCTGAGTACT-3'; downstream
5'-TGACAATGAGGACCCAGCCTGCAA-3' according to (30). Two sets of primers
for the NPR-B based on the published rat gene sequence (31) were used:
1) upstream 5'-AACGGGCGCATTGTGT-ATATCTGCGGC-3'; downstream
5'-TTATCACAGGATGGGTCGTCCAAGTCA-3' according to (30); 2) according to
(32) upstream: 5'-AACTGATGCTGGAGAAGGAGC-3'; downstream
5'-TACTCCGG-GTGACGATGCAGAT-3'. Nucleotides for the mouse NPR-C gene
sequence (33) were designed as follows upstream:
5'-CTACATCCAAGGCAGCGAGCG-3'; downstream: 5'-GCAACCACAGAGAAGTCCCCA-3'.
The PCR products were expected to have the following sizes: NPR-A 451
bp; NPR-B 692 bp and 355/280 bp, respectively; NPR-C:492 bp.
PCR amplification method.
PCR was performed in principle as
described previously (18). Amplification products were radiolabeled by
adding (
-32P)dCTP (1 µCi) to each reaction sample.
NPR-A receptor transcripts were amplified in 30 cycles of annealing (55
C, 1 min), extension (73 C, 2 min) and denaturation (93 C, 1 min).
NPR-B transcripts using the oligonucleotides described in (30) were
amplified (28 cycles) as follows: annealing (60 C, 1 min), extension
(73 C, 2 min), denaturation (93 C, 1 min). Using the oligonucleotides
described by (32), the amplification was performed as described in (32)
and PCR-transcripts have not been radiolabeled. The PCR conditions for
the NPR-C were the following: annealing (53 C, 1.5 min), extension (72
C, 3 min), denaturation (93 C, 1 min), 35 cycles.
To control for unspecific amplification, a sample containing no cDNA or not reverse transcribed macrophage mRNA was employed in each PCR experiment. As positive controls, either cDNA extracted from mouse ventricle or kidney known to possess all three NPR (30) were subjected to the corresponding PCR. Aliquots of PCR products were submitted to gel electrophoresis (PAGE, 6% polyacrylamide) and NPR transcripts were identified by silver nitrate staining followed by exposure to x-ray films (-70 C, 18 h, Hyperfilm MP, Amersham, Braunschweig, Germany). NPR-B products obtained with the primer pair described in (32) were separated by agarose electrophoresis (2% agarose) and stained by ethidium bromide.
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Results |
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). Macrophages were stimulated
with LPS (1 µg/ml) for 20 h to evoke NO synthesis, which was
measured by determining the concentration of nitrite in the
supernatant. Coincubation of all four types of macrophages with ANP
(10-10–10-6 M) and LPS (1
µg/ml) resulted in a dose-dependent reduction of nitrite
accumulation. In each type of LPS-activated cells, ANP concentrations
up to 10-8 M significantly inhibited
NO-production. ANP (10-6 M) in the absence of
LPS did not alter the basal nitrite accumulation (data not shown). The
degree of NO-suppression by ANP (10-6 M)
differed depending on the type of macrophage employed. ANP displayed a
more pronounced NO inhibitory effect, i.e. an up to 90%
reduction of nitrite accumulation in macrophage cell lines as compared
with the primary macrophages (up to 60% and 50% for BMM and PM,
respectively). BMM and PM display a higher LPS-induced NO production as
compared with the cell lines (nitrite accumulation per mg cell protein:
BMM >PM >RAW 264.7 >J 774; data not shown). The basal level of
nitrite accumulation in the supernatant of PM was significantly higher
than in the other cells types (Fig. 1).
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Effect of other NP on NO-synthesis of BMM
To elucidate whether the NO-inhibitory effect is specific for ANP
99–126 we examined the ANP fragment, ANP 102–123 (atriopeptin I, AP
I) and urodilatin, which both differ in their amino acid sequence from
ANP, but bind to the same receptor (NPR-A). Furthermore, the ligand for
the other guanylate cyclase coupled receptor (NPR-B), CNP, was
employed. As shown in Fig. 2, atriopeptin
I (A) as well as urodilatin (B) reduced nitrite accumulation of LPS
activated BMM. However, API and urodilatin were only effective at a
concentration of 10-6 M and elicited only a
15% (AP I) and 20% (urodilatin) decrease in NO-production,
respectively. In contrast, CNP even at the high concentration of
10-5 M did not affect the nitrite formation of
LPS-activated BMM (Fig. 2C).
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Expression of NPR-mRNA in macrophages
PCR amplification products of mRNA coding for NPR-A, NPR-B, and
NPR-C were detected in BMM as demonstrated in Fig. 3A. Size of PCR products were estimated
by comparison with a size marker and found to correspond to the
calculated ones of 451 bp for the NPR-A, 692 for the NPR-B, and 492 bp
for the NPR-C. Furthermore, NPR transcripts of macrophages comigrated
with amplification products of either ventricle or kidney of mice known
to express all three NPR (30). Because it has been reported that two
different forms of NPR-B exist that differ in their functional activity
(32), a specific set of primers that allows for their simultaneous
detection was used for RT-PCR. We found that BMM contain predominantly
the functional active NPR-B1 (355 bp transcript) and to a lesser degree
the nonfunctional NPR-B2 (280 bp PCR product). Similar PCR products
were obtained from kidney cDNA employed as positive control.
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shows representative
HPLC-profiles of radiolabeled ANP (upper panel) and CNP
(lower panel) immediately after added to the LPS exposed BMM
(0 h) and after 20 h incubation. The peak of the intact peptide,
which is prominent at time 0, declined considerably after 20 h
incubation, whereas a second peak (Vo), which most likely
represents smaller radiolabeled fragments, largely increased. No
significant difference in the HPLC profiles were observed when
untreated instead of LPS-activated BMM cells were employed (data not
shown).
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, the percentage of intact peptide was plotted against the
duration of incubation. No significant difference of the stability of
ANP and CNP was observed in LPS treated BMM. CNP, however, seemed to be
faster degraded than ANP when exposed to LPS activated RAW 264.7 and J
774 cells. Inhibition of neutral endopeptidase (EC 3.4.24.11), an
enzyme important for degradation of NP, by phosphoramidon and
thiorphan, as well as by a combination of both did not significantly
prevent the degradation (data not shown).
Receptor selectivity of the ANP effect on NO synthesis
To determine which NP-receptor mediates the inhibitory effect of
ANP on NO synthesis the following experiments were performed with
LPS-stimulated cells. As shown in Fig. 5A, stable analogues of cGMP,
i.e. 8-Br-cGMP and dibutyryl-cGMP at a concentration of
10-3–10-5 M reduced nitrite
accumulation (up to 60%). Furthermore, an antagonist of the guanylate
cyclase coupled NPR, HS-142–1, dose dependently (1, 10, 100 µg/ml)
abrogated the NO reducing effect of ANP (10-6 or
10-7 M) in BMM (Fig. 5B). LY 83583
(10-6–10-8 M), a compound known
to inhibit cGMP production, partly abolished the reduction of nitrite
accumulation by ANP (10-6 M) (Fig. 5C). The
specific NPR-C ligand, C-ANF did not elicit a significant decrease in
nitrite concentration even at a concentration as high as
10-5 M (Fig. 5D). Similar results were
obtained when employing RAW 264.7 as well as J 774 cells (data not
shown). ANP, the cGMP analoga, HS-142–1, LY 83583 as well as C-ANF
were tested for their effect on NO synthesis in untreated cells and did
not exhibit any effect (data not shown).
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shows that reduction of nitrite
accumulation (24 h) by ANP as well as by dexamethasone was highest when
added simultaneously with LPS. ANP and dexamethasone were ineffective
when given 12 h after LPS stimulation, whereas nitrite
accumulation of L-NMMA exposed cells was reduced by 50%.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The induction of nitric oxide synthesis in many cell types has been identified as part of the host response to sepsis and inflammation (for review see Refs. 34–37). NO can be detrimental as well as beneficial during inflammation depending on the amount, duration, and cellular site of production. Therefore, special interest focuses on the regulatory mechanism of NO production and on tools for potential pharmacological intervention with the responsible enzyme, i.e. the inducible NO-synthase (iNOS) (34, 36, 37). In this respect, macrophages as the prominent cells of the immune system expressing iNOS, have been thoroughly studied (36, 37). RAW 264.7 cells that have been used to purify and clone the mouse iNOS (38), as well as J774 cells (39), are the most frequently used cell models for studies on iNOS. In addition, various studies were performed on mouse primary macrophages such as peritoneal and bone marrow derived macrophages (40, 41). It has been suggested that the various types of macrophages might differ in the degree of iNOS activation as well as in their subcellular distribution of iNOS and moreover in yet undocumented differences in their posttranslational mechanism regulating iNOS (40, 41, 42). Therefore, we considered it as important to compare our previous finding of the ANP-effect on NO synthesis in primary mouse macrophages to the cell lines RAW 264.7 and J 774. We observed that ANP inhibited NO production in all four types of macrophages. This finding corroborates a new aspect of ANP bioactivity. The cell-dependent differences of the extent of inhibition by ANP may indeed reflect variations in the iNOS system between different types of macrophages.
The effect seems to be specific for ANP because urodilatin and atriopeptin I induced only a slight decrease of NO synthesis and importantly, CNP displayed no activity. The lack of CNP action cannot be attributed to the fact that receptors for the NP are missing on the macrophages used. Our data demonstrate for the first time, that macrophages express mRNA coding for all three receptor subtypes, i.e. NPR-A, NPR-B, and NPR-C. Some evidence for the existence of the NPR-A has been given in the Lature before as elevated cGMP accumulation could be monitored for J774 and human peritoneal macrophages after exposure to ANP (43, 44).
Urodilatin has receptor binding properties similar to ANP (45). However, there are some reports on divergent biological actions of the two NP. The effects of urodilatin on the systemic blood pressure for instance are less pronounced compared with ANP (46). On the other hand, urodilatin protects against bronchoconstriction, whereas ANP had no effect (47). Our data provide information on another biological system in which ANP and urodilatin act differently.
We showed that macrophages express the NPR-B and thus should be target cells for CNP action. CNP had no effect on the NO production of macrophages. Certainly, CNP may just interfere with functional parameters of macrophages not tested. In accordance with our data, however, others also reported that CNP elicits very low or no biological activity despite the presence of the CNP-specific receptor (NPR-B), (12, 14, 15, 16, 17). Thus, the lack of action of CNP in our assay system may deserve a more thorough discussion for various reasons. Firstly, CNP has shown to have a higher turnover than ANP in vivo as well as in other cell systems (48, 18). In fact, degradation of CNP in RAW 264.7 and J 774 cells was higher as compared with that of ANP, but no significant difference was observed when BMM were employed. Because CNP even at 10-5 M was without effect and ANP was active at least over a three log range of concentration, it is very unlikely that differences in stability account for the lack of effect of CNP.
A further point of discussion should be the heterogenity of the NPR-B receptor. Two forms of NPR-B receptor have been detected in a variety of tissues and shown to differ from each other only by a 75 bp deletion at the 3' flanking region (32). The two forms of NPR-B possess practically the same high binding affinity for CNP; however, the shorter form could not induce cGMP production upon binding by CNP (32). We could detect both forms of NPR-B transcripts in the macrophages used. Because the ANP effect on NO synthesis is shown to be mediated by cGMP, an insufficient amount of cGMP produced by CNP may indeed be responsible for the lack of NO inhibition by this peptide.
The fact that the ANP effect is mediated via the NPR-A receptor was conclusively demonstrated as both 8-Br-cGMP and dibutyryl-cGMP dose dependently mimic the ANP effect and employment of the microbial polysaccharide HS-142–1, which selectively blocks the guanylate cyclase-linked NP receptors and cGMP production (23), dose dependently reverses the ANP effect. Because no soluble guanylate cyclase has been detected in the macrophages employed (49, our unpublished observation) the reversal of the ANP effect by LY 83583 can be attributed to an inhibition of cGMP production linked to the particulate guanylate cyclase-linked NPR-A receptor. Finally, the fact that AP I shows a very weak effect on NO synthesis also argues for a cGMP mediated ANP effect because deletion of amino acids from the carboxyl and/or amino terminal of ANP has shown to markedly diminish the ability to increase cGMP (3).
The inhibition of NO synthesis via cGMP seems to be a function of the cell type. It is known that release of NO results in stimulation of soluble guanylate cyclase leading to a profound increase in cGMP within target cells such as vascular smooth muscle cells (35, 36). cGMP is considered to be the major second messenger of NO with respect to its physiological as well as pathophysiological effects on the vascular tone. In contrast to our findings in macrophages, cGMP as well as atrial natriuretic peptide stimulate NO synthesis in vascular smooth muscle cells (50, 51). Thus, the ANP induced inhibition of NO in macrophages might implicate a specific role of the peptide in immunological mechanism mediated by macrophages.
The mechanism of the NO inhibitory effect of ANP in macrophages could be either an interaction directly with the enzyme activity or with transcriptional and/or translational processes of the iNOS. Experiments employing dexamethasone, a known inhibitor of iNOS induction and L-NMMA, an inhibitor of iNOS activity (34) in comparison to ANP revealed that the inhibition of nitrite accumulation over 24 h by dexamethasone as well as ANP decreased when given 2–8 h after LPS. Interpreting these data, one has to take into account that LPS-stimulated macrophages may already produce considerable levels of NO during that period of time. Thus, changes in either enzymatic activity or expression of iNOS by ANP based on this observation have to be discussed with caution. Nevertheless, it seems not very likely that ANP acts as classical inhibitor of iNOS activity because L-NMMA still decreased nitrite accumulation when added to cells 12 h after LPS, whereas ANP as well as dexamethasone were ineffective. Experiments examining enzyme activity and iNOS are planned to clarify the mechanism of action of ANP.
In summary, our data suggest a specific role for ANP, namely an interference with the macrophage iNOS system via the NPR-A receptor.
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Acknowledgments |
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Footnotes |
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Received May 19, 1997.
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