0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society
Vol. 138, No. 10 Printed in U.S.A.
Effects of Different Natriuretic Peptides on Nitric Oxide Synthesis in Macrophages* ALEXANDRA K. KIEMER
AND
ANGELIKA M. VOLLMAR
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Ko¨niginstrasse 16, D-80539 Munich, Germany ABSTRACT Atrial natriuretic peptide (ANP) has previously been suggested to inhibit the production of NO in LPS-activated primary macrophages. The aim of the present study was 1) to examine whether ANP elicits this effect also on macrophage cell lines (RAW 264.7, J774), 2) to elucidate whether ANP is the only natriuretic peptide (NP) inhibiting NO synthesis, 3) to look for the expression of natriuretic peptide receptors (NPR) on macrophages, 4) to consequently determine the type of receptor mediating the ANP effect and 5) to obtain first information on the underlying mechanism. Whereas ANP dose dependently (1026–1028 M) inhibited NO synthesis (measured as nitrite accumulation, 20h) in all four types of macrophages (bone marrow derived and peritoneal macrophages; RAW 264.7 and J 774), urodilatin and atriopeptin I displayed only a weak effect restricted to the highest concentration (1026 M) employed. Importantly, C-type natri-
uretic peptide (CNP) showed no NO-inhibitory effect. The lack of effect of CNP was shown not to be due to its lower stability or its missing receptor. Macrophages were shown to express all three natriuretic peptide receptors (NPR-A, NPR-B, NPR-C) using RT-PCR technique. Furthermore, two types of NPR-B seem to be present in macrophages. The effect of ANP was mediated via the guanylate cyclase coupled NPR-A as shown by experiments employing stable cGMP analogs, the NPR-A antagonist HS-142–1, LY-83583, a cGMP inhibitor as well as C-ANF, a specific ligand of the NPR-C. Reduction of nitrite accumulation by ANP was highest when added simultaneously with LPS and abolished when added 12 h after LPS stimulation. In summary, ANP was shown to inhibit NO production of LPS-activated macrophages via cGMP. (Endocrinology 138: 4282– 4290, 1997)
T
imental conditions are rare. There is some evidence that the expression of NP within one tissue is differentially regulated as shown for the heart, the gut, or macrophages (9 –11). Furthermore, information on the bioactivity of the NP in conscious sheep as well as their effects on water intake and cell growth exists (12–14). In addition, different distribution and regulation of NP-receptors have been demonstrated (11, 15–18). 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
HE MEMBERS of the natriuretic peptide family, atrial natriuretic peptide (ANP) and urodilatin, brain natriuretic peptide (BNP) as well as C-type natriuretic peptide (CNP), possess a high degree of sequence homology, especially within the 17-amino acid ring formed between two cysteine residues (for review see Refs. 1 and 2). This ring structure seems to be important for binding to guanylate cyclase coupled natriuretic peptide receptors (NPR) (3). ANP, urodilatin, and BNP activate the A-type receptor (NPR-A), whereas CNP is the specific ligand for the B-type receptor (NPR-B) (4). All natriuretic peptides bind to the nonguanylate cyclase-linked C-type receptor (NPR-C), which is known for its clearance function (5). ANP and BNP are circulating peptides of predominantly cardiac origin and have shown to share functional homology as both are diuretic, natriuretic and vasodilating (1, 2). Urodilatin closely relates to ANP and was isolated from human urine (6). It differs from ANP by four amino acids at the N-terminus of the peptide, and so far little is known about its physiological function (1, 6). CNP is suggested to be the major natriuretic peptide in the brain (7) but was also found in peripheral cells such as endothelial cells (8) and macrophages (9). Compared with ANP, CNP has insignificant natriuretic and diuretic but distinct vasoactive properties (7, 8). Thus, CNP may play a role quite different from ANP and BNP. However, comparative studies on ANP/BNP vs. CNP using the same experReceived May 19, 1997. Address all correspondence and requests for reprints to: Angelika M. Vollmar, Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Ko¨niginstrasse 16, D-80539 Munich, Germany. E-mail:
[email protected]. * This work was supported by the DFG (Vo 376/8 –1).
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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 NOinhibition by ANP was obtained. Materials and Methods Materials ANP 99 –126 was purchased from Calbiochem/Novabiochem (Bad Soden, Germany); CNP-22, urodilatin and atriopeptin I (ANP 102–123) were from Saxon Biochemicals (Hannover, Germany). 125I-ANP (specific activity 1000 Ci/mmol) and 125I-CNP (specific activity 2000 Ci/mmol) were purchased from Peninsula (Merseyside, UK). HS-142–1 was a gift from Dr. Matsuda, Tokyo Research Laboratories (Tokyo, Japan). 8-BrcGMP, dibutyryl-cGMP and LY 83583 were obtained from Sigma (Deisenhofen, Germany). All other products were either from Sigma or ICN Biomedicals (Eschwege, Germany).
Cell culture
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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 mg/ml) macrophages were employed. Furthermore, degradation of NP was determined in LPS-activated cells pretreated (1 h) with thiorphan (1025 m) or phosphoramidon (1025 m), or the combination of both. Supernatants (n 5 3) were collected at various times after addition of NP and analyzed by reverse phase-HPLC (C18 Bondapak column; 4.5 3 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
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.
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 mg) was transcribed to cDNA using avian myeloblastosis virus reverse transcriptase (Promega) according to established protocols (9, 25).
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 mg/ml). 2 3 105 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 3 106 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).
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: 59-AAGAACCCGATAATCCTGAGTACT-39; downstream 59-TGACAATGAGGACCCAGCCTGCAA-39 according to (30). Two sets of primers for the NPR-B based on the published rat gene sequence (31) were used: 1) upstream 59-AACGGGCGCATTGTGTATATCTGCGGC-39; downstream 59-TTATCACAGGATGGGTCGTCCAAGTCA-39 according to (30); 2) according to (32) upstream: 59AACTGATGCTGGAGAAGGAGC-39; downstream 59-TACTCCGGGTGACGATGCAGAT-39. Nucleotides for the mouse NPR-C gene sequence (33) were designed as follows upstream: 59-CTACATCCAAGGCAGCGAGCG-39; downstream: 59-GCAACCACAGAGAAGTCCCCA-39. 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.
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 mg/ml) in the presence or absence of various concentrations of NP or other indicated substances. Test substances were dissolved in medium and stored (270 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 ml of supernatant was removed and 90 ml 1% sulfanilamide in 5% H3PO4 and 90 ml 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.
PCR amplification method. PCR was performed in principle as described previously (18). Amplification products were radiolabeled by adding (a-32P)dCTP (1 mCi) 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 expo-
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sure to x-ray films (270 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.
Results Effect of ANP on NO synthesis of various types of macrophages
The effect of ANP on NO synthesis in primary macrophages (BMM, PM) should be compared with the macrophage cell lines (RAW 264.7; J 774) (Fig. 1). Macrophages were stimulated with LPS (1 mg/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 (10210–1026 m) and LPS (1 mg/ml) resulted in a dose-dependent reduction of nitrite accumulation. In each type of LPS-activated cells, ANP concentrations up to 1028 m significantly inhibited NO-production. ANP (1026 m) in the absence of LPS did not alter the basal nitrite accumulation (data not shown). The degree of NO-suppression by ANP (1026 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
FIG. 1. ANP dose dependently inhibits the formation of nitrite in BMM, PM, RAW 264.7, and J 774 cells, respectively. Cells were cultured for 20 h in either medium alone (Co) or medium containing LPS (1 mg/ml) or a combination of LPS (1 mg/ml) and various concentrations of ANP (10210–1026 M). Culture supernatants were assayed for nitrite accumulation using the Griess reaction (Materials and Methods). Data are expressed as percentage of nitrite concentration accumulated in the supernatant of LPS-activated macrophages (100%) and represent the mean 6 SEM of n 5 3 wells of four to six independent experiments. *, P , 0.05 represent significant differences compared with the values seen in LPS activated cells (two-tailed Welch test).
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with the primary macrophages (up to 60% and 50% for BMM and PM, respectively). BMM and PM display a higher LPSinduced 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). Cytotoxicity of ANP
The inhibitory effect of ANP on NO synthesis was not due to cytotoxicity of ANP. As an indicator of cell viability, the mitochondrial activity of the cells was assessed for each experiment performed. Cells exposed to LPS (1 mg/ml, 20 h) showed an about 10 to 30% (depending on the cell type) reduction of mitochondrial respiration compared with untreated cells; however, no difference between LPS (1 mg/ml) and LPS and ANP (1026–10210 m) treated cells could be detected (less than 10% difference in each experiment). 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
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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 1026 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 1025 m did not affect the nitrite formation of LPS-activated BMM (Fig. 2C). Similar results were obtained employing the macrophage
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cell lines RAW 264.7 and J 774. The NP had no effect on basal NO production, and no cytotoxicity could be demonstrated using the MTT test (data not shown). 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. Stability of ANP and CNP in macrophages
The lack of effect of CNP on NO synthesis could be due to its higher degradation rate compared with ANP. Therefore, the percentage of intact radiolabeled ANP and CNP incubated for 5, 10, 15, and 20 h, respectively, with untreated as well as LPS stimulated macrophages was examined. Figure 4A 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). In Fig. 4B, 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 FIG. 2. Effect of atriopeptin I, urodilatin, and CNP on NO synthesis by LPS activated BMM. Nitrite accumulation in the supernatant of either untreated cells (Co) or cells treated with LPS (1 mg/ml) and cells costimulated with LPS and atriopeptin I (API, panel A), urodilatin (uro, panel B) and CNP (panel C), respectively, is shown. Peptides were coincubated with LPS at concentration of 1026–1028 M (API and urodilatin) and 1025–1027 M (CNP). Results are expressed as percentage of nitrite accumulation in the supernatant of LPS-treated cells and represent the means 6 SEM of three (API, urodilatin) and six (CNP) independent experiments performed in triplicates. *, P , 0.05 refers to LPS treatment (two-tailed Welch test).
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 dibutyrylcGMP at a concentration of 1023–1025 m reduced nitrite accumulation (up to 60%). Furthermore, an antagonist of the guanylate cyclase coupled NPR, HS-142–1, dose dependently (1, 10, 100 mg/ml) abrogated the NO reducing effect of ANP (1026 or 1027 m) in BMM (Fig. 5B). LY 83583 (1026–
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FIG. 3. Representative PCR experiment demonstrating the presence of mRNA coding for NPR-A, NPR-B and NPR-C in BMM. Poly(A)1 RNAs were isolated from BMM as well as from mouse ventricle and kidney (positive control) and amplified by RT-PCR as described in Material and Methods. A, 32P-labeled amplification products were size fractionated by PAGE and exposed to x-ray film. NPR-A transcripts (451 bp): lane 1 ventricle cDNA (10 ng), lane 2 BMM cDNA 100 ng. NPR-B transcripts (692 bp): lane 1 ventricle cDNA (10 ng), lane 2 BMM cDNA (100 ng). NPR-C transcripts (492 bp): lane 1 kidney cDNA (100 ng) lane 2 BMM cDNA (100ng). B, Detection of the two forms of NPR-B in BMM. PCR with the pair of primer for the NPR-B sequence described in (32) yields two NPR-B specific DNA fragments of 355 bp and 280 bp, respectively. After electrophoresis (2% agarose) the corresponding PCR transcripts were stained by ethidium bromide. Lane 1 cDNA from kidney (positive control, 50 ng); lane 2 cDNA from BMM (200 ng).
1028 m), a compound known to inhibit cGMP production, partly abolished the reduction of nitrite accumulation by ANP (1026 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 1025 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, HS142–1, 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). Time dependency of the ANP effect
ANP (1026 m) was added either simultaneously with LPS or 2, 4, 8, and 12 h later. In addition to ANP, dexamethasone (1026 m) known to inhibit iNOS induction and NG-monomethyl-l-arginine (L-NMMA, 1023 m), an inhibitor of the catalytic activity of the NO-synthase (34), were employed. Figure 6 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%. Discussion
The study reported here demonstrates that atrial natriuretic peptide selectively decreases the NO production by macrophages via the guanylate-cyclase coupled NPR-A receptor. 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 – 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
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FIG. 4. Degradation of ANP and CNP incubated with LPS-activated macrophages. Panel A shows representative RP-HPLC profiles of either 125 I-ANP (10 nM, upper chromatogram) or 125I-CNP (10 nM, lower chromatogram) added to LPS-treated BMM for 0 (solid line) and 20 h (dotted line), respectively. Elution position of intact radiolabeled peptides was marked by arrows and was determined by employing freshly purchased radiolabeled NP. Radioactivity eluted at the void volume (Vo) of the column refers to degraded material. B, Time dependency of ANP and CNP degradation in LPS-activated BMM, J 774, and RAW 264.7 cells, respectively. The amount of intact peptide at 0 h incubation time was defined 100%. Data given represent percentage of recovery of intact peptide (ANP, solid line; CNP, dotted line) blotted against the period of incubation with the macrophages. Each point represents the mean (6 SEM) of two independent experiments.
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 –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 1025 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
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FIG. 5. Characterization of the NPR responsible for the ANP induced NO-inhibition in macrophages (BMM). Cells were treated with LPS (1 mg/ml, 20 h), nitrite accumulation was measured (Griess assay) and referred to 100%. A, Effect of 8-Br-GMP (BrcGMP) and dibutyryl-GMP (dibcGMP) both at 1023–1025 M. B, Effect of coincubation of increasing amounts of the NPR-A antagonist, HS-142–1 (1–100 mg/ml) with ANP (1026 and 1027 M, respectively). C, Effect of coincubation of ANP (1026 M) and LY 83583 (1028–1026 M), an antagonist to cGMP generation. D, Effect of the NPR-C specific ligand, C-ANF (1028–1025 M). Representative experiments out of three similar ones are shown. Bars represent the mean 6 SEM of triplicates. Data are expressed as percentage of nitrite concentration found in LPS-activated cells after 20 h cultivation (100%, open bar).
each other only by a 75 bp deletion at the 39 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 HS142–1, 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
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References
FIG. 6. Time dependency of inhibition of nitrite accumulation by ANP. ANP (1026 M, filled bars), dexamethasone (1026 M, open bars) and L-NMMA (1023 M, striped bars) were added either simultaneously (0 h) with LPS (1 mg/ml) or 2, 4, 8, and 12 h after LPS stimulation. Nitrite accumulation in the supernatant was measured 20 h after LPS stimulation by the Griess reaction as described in Materials and Methods. Data are expressed as percentage of nitrite concentration in supernatant of only LPS-treated cells (100%) and represent the mean 6 SEM of three experiments performed in triplicates. *, P , 0.05 refers to LPS treatment (two-tailed Welch test).
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. Acknowledgments We like to thank Ursula Ru¨berg for her excellent technical assistance and Dr. R. Schulz (Munich) for helpful discussions. We are grateful to Dr. Matsuda, Tokyo, for providing the polysaccharide HS-142–1.
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