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(Journal of Leukocyte Biology. 2003;73:502-510.)
© 2003 by Society for Leukocyte Biology

Differential sensitivity of human monocytes and macrophages to ANP: a role of intracellular pH on reactive oxygen species production through the phospholipase involvement

P. M. Baldini, P. De Vito, A. Martino, M. Fraziano, C. Grimaldi, P. Luly, F. Zalfa and V. Colizzi

Department of Biology, University of Rome "Tor Vergata," Italy

Correspondence: Dr. P. M. Baldini, Department of Biology, University of Rome "Tor Vergata," Via della Ricerca Scientifica, 00133 Rome, Italy. E-mail: Baldini{at}uniroma2.it


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ABSTRACT
 
Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present work was to study the effect of ANP on the intracellular pH (pHi) of human monocytes and macrophages and to investigate whether pHi changes could play a role on phospholipase activities and reactive oxygen species (ROS) production. Human macrophages isolated by peripheral blood mononuclear cells and THP-1 monocytes, which were shown to express all three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C), were treated with physiological concentrations of ANP. A significant decrease of pHi was observed in ANP-treated macrophages with respect to untreated cells; this effect was paralleled by enhanced phospholipase activity and ROS production. Moreover, all assessed ANP effects seem to be mediated by the NPR-C. In contrast, no significant effect on pHi was observed in THP-1 monocytes treated with ANP. Treatment of macrophages or THP-1 monocytes with 5-(N-ethyl-N-isopropyl)amiloride, a specific Na+/H+ antiport inhibitor, decreases pHi in macrophages and monocytes. Our results indicate that only macrophages respond to ANP in terms of pHi and ROS production, through diacylglycerol and phosphatidic acid involvement, pointing to ANP as a new modulator of ROS production in macrophages.

Key Words: phosphatidic acid • diacyglycerol • NADPH oxidase


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INTRODUCTION
 
Atrial natriuretic peptide (ANP) is a polypeptide hormone mainly secreted by the heart atria in response to atrial stretch, able to induce natriuresis and vasodilatation "in vivo" with subsequent regulation of blood pressure homeostasis [1 ]. ANP promotes its biological effects mainly through two biochemically and functionally distinct classes of ANP receptors located on the plasma membrane of target cells: type natriuretic peptide receptor (NPR)-A, which activates a particulate guanylate cyclase, giving rise to guanosine 3',5'-cyclic monophosphate (cGMP), and NPR-C, which regulates adenylate cyclase and the adenosine 3',5'-cyclic monophosphate system and/or membrane lipid turnover through the activation of specific phospholipases (PLs) [2 ]. The functions of ANP, however, are not only restricted to blood pressure homeostasis [3 ] but also seem to play an important role in the immune system, as thymus [4 ] and macrophages [5 ] are sites of synthesis of the hormone. In particular, ANP was found to inhibit maturation and differentiation of fetal thymus [6 ], as well as proliferation of thymocytes of adult animals [4 ]. Recently, Matsuo [7 ] showed that the monocytic cell line THP-1 is able to produce C-type NP when differentiated in macrophages after treatment with phorbol ester, but the mechanism involved in monocytes/macrophages differentiation as well as the sensitivity to ANP in undifferentiated monocytes have not been completely defined.

Phagocytic cells represent the first line of defense against intracellular pathogens [8 ], and increasing evidence suggests an involvement of PLD activation in THP-1 cells [9 ]. It has been reported that phagocyte activation in response to extracellular agonists by different classes of effector enzymes, including PLs, induces degranulation [10 ], phagocytosis [11 ] and respiratory burst [12 ]. In particular, PLD is able to hydrolyze phosphatidylcholine (PC), producing choline and phosphatidic acid (PA), which in turn, can be metabolized further into bioactive lipids such as diacylglycerol (DAG), a known activator of protein kinase C (PKC), which is also produced by a PC-dependent PLC (PC–PLC) pathway [13 ]. It is well known that PA, in phagocyte cells, can play an important role as a second messenger for its direct role in secretory vesicle budding [14 ] and in the activation of the respiratory burst, also being able to activate reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme responsible for reactive oxygen species (ROS) production [12 ]. It is interesting that ANP stimulates production of reactive oxygen in macrophages [15 ] and is able to inhibit the production of nitric oxide as well as of nuclear factor-{kappa}B, the trascription factor responsible for tumor necrosis factor {alpha} expression and secretion in macrophages activated by lipopolysaccharide [16 ].

Changes in cytoplasmic pH [intracellular pH (pHi)] in response to a variety of ligands may act as a signaling event for the regulation of neutrophil degranulation. In fact, Gewirtz et al. [17 ] showed that neutrophil degranulation and PLD activation are enhanced if the Na+/H+ antiport is blocked by amiloride and its analogs such as 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Strictly connected to the previously mentioned pHi is the role of the Na+/H+ antiport: a plasma membrane protein that exchanges sodium and hydrogen ions according to their concentration gradient, whose main function is the regulation of pHi and cell volume [18 ]. ANP may inhibit or stimulate the Na+/H+antiport depending on cell type: It inhibits the Na+/H+ antiport in rat brain synaptosomes [19 ] and chicken villus enterocytes [20 ], and it acts as a stimulator in rat aortic smooth muscle cells [21 ].

The aim of the present study was to assess the effect of ANP or EIPA treatment on pHi changes, respectively, in human primary macrophages and in human monocytoid cell line THP-1; to establish whether the inhibition of the Na+/H+ antiport in macrophages may be responsible for the activation of PLs and ROS production; and to investigate which NPR mediates the ANP-induced effects in human macrophages. We report that macrophages but not THP-1 monocytes are sensitive to ANP treatment and that blocking the Na+/H+ antiport followed by intracellular acidification represents an early event for DAG and PA production as related to PC–PLC and PLD involvement and ROS generation.


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MATERIALS AND METHODS
 
Primary cell culture
Peripheral blood mononuclear cells were isolated from human buffy-coat and monocytes purified by 1 h adherence on T75 tissue-culture flasks (Corning, Cambridge, MA) [22 ]. Thereafter, nonadherent cells were removed by two washes with warm RPMI 1640, and adherent monocytes were collected by gentle scraping with a cell scraper (Sarstedt, Newton, NC) after 15 min incubation with 5 mM EDTA in phosphate-buffered saline at 4°C. Cells were then suspended at 106/mL in complete medium [RPMI 1640, supplemented with 10% fetal calf serum (FCS), lowered to 0.5% before each experiment, 5 mM L-glutamine, and 5 µg/ml gentamicine] and were cultured for a further 7–10 days in polystyrene 24-well plates to obtain differentiated monocyte-derived macrophages. The purity of such population was assessed by flow cytometry, always being not less than 80%.

Cell culture
The human monocytic cell line THP-1 was obtained from American Type Culture Collection (Manassas, VA), cells were cultured in Falcon flasks, with RPMI-1640 medium supplemented with 5% FCS, lowered to 0.5% before each experiment, 2 mM L-glutamine, 100 µg/ml streptomycin, penicillin (100 U/ml), 0.25% glucose, 110 µg/ml sodium pyruvate, and maintained at 37°C in a humidified atmosphere containing 5% CO2.

pH Measurements
Cytoplasmatic pH was measured by fluorescence spectrometry using the intracellular probe 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein/acetoxymethylester (BCECF/AM) as described previously [23 , 24 ]. To rule out the contribution of HCO3--dependent transport mechanisms, all experiments were performed in HCO3--free buffer with the following composition (mM): 135 NaCl, 1 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 20 Hepes, pH 7.4. This buffer (designated as Na+ buffer) was used for incubation with the fluorescent probe and for the determination of pHi unless otherwise stated; cells incubated in this buffer were considered virtually depleted of HCO3-. Incubation with the fluorescent dye was performed as follows: Cells were washed twice with Na+ buffer and were thus considered HCO3--free; next, cells were incubated in Na+ buffer with the fluorescent dye [1 mg/ml in dimethyl sulfoxide (DMSO)] at the final concentration of 1 µg/ml for 30 min at 37°C in the dark. After incubation, the medium containing the dye was discarded, and the cells were washed two times with the same buffer. In experiments with choline buffer, external Na+ was replaced by equimolar choline chloride concentration to keep quiescent the antiport. The calibration curve was performed as reported previously [25 ] using the nigericin method in high-potassium medium with the same composition as the Na+ buffer, but equimolar KCl substituted for NaCl. The calibration curve was linear in the pH range, 6.5–7.5 (not shown). Fluorescence was measured under continuous magnetic stirring at a controlled temperature (37°C) in a Perkin–Elmer LS-5 luminescence spectrometer equipped with a chart recorder Model R 100A, with excitation and emission wavelengths of 500 nm and 530 nm, using 5 and 10 nm slits, respectively, for the two light paths. Fluorescence was also routinely measured at 450 nm excitation (at this wavelength, the fluorescence is proportional to intracellular dye concentration but is relatively pH-insensitive), and the value did not change more than 10% during the experimental period. The block of the Na+/H+ antiport was obtained with the specific inhibitor EIPA, and the cellular mortality was evaluated by trypan blue staining using a Neubauer-modified chamber.

Radioactive labeling and treatment
Preliminary experiments on [3H]-myristate incorporation indicated that optimal labeling was at 3 h (1 µCi/ml). After labeling, the cells were left for 1 h in the medium without FCS and then were incubated in Na+ or choline buffer and treated with EIPA, ANP (99–126), or the ring deleted analog of ANP (cANF) (4–23) for different experimental times. Incubation was stopped with 1 ml ice-cold methanol. When requested, cells were pretreated for 1 h with 5 µM tryciclodecan-9-yl-xanthogenase (D609), 10-7 M calphostin-c, or 5 µM 3-1-3-(amidinothio)propyl-1H-indol-3-(methyl-1H-indol-3-yl)maleimide methane sulfonate (Ro-31-8220).

Thin-layer chromatography of lipids
Cells were scraped off, and lipids were extracted by the method of Bligh and Dyer [26 ]. The dried lipids were redissolved in 20 µl CH3Cl. DAG was separated by chromatography on 10 x 10 cm silica gel-coated glass plates and activated at 100°C for 1 h immediately before use. The solvent consisted of light petroleum [base pair (bp) 40–60°C)/diethyl ether/acetic acid (80/20/1 v/v). After chromatography, plates were dried under N2, and the spots revealed under iodine vapor were scraped off, eluted with 0.2 ml ethanol, and counted for radioactivity after the addition of 7 ml Optifluor (Packard Instruments, Downers Grove, IL). Lipids were identified by comparing their retention factor (Rf) values with those of authentic standards obtained from Supelco (Bellefonte, PA).

Transphosphatidylation reaction
To determine PLD activity [27 ], 1% ethanol was added to prelabeled cells 15 min before the addition of ANP or EIPA. PA and phosphatidylethanol (PE) were separated by thin-layer chromatography on 10 x 20 cm silica gel 60 plates. The solvent system consisted of ethyl acetate/iso-octane/acetic acid/H2O (130/20/30/100, v/v). PE was identified by comparing its Rf value with that of a standard according to Chattopadhyay et al. [28 ].

RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from human THP-1 monocytes or macrophages (~2x106 cells) by the proteinase K–sodium dodecyl sulfate method [29 ]. RNA samples were quantified by measuring UV absorbance at 260 nm using a BioPhotometer (Eppendorf, Hamburg, Germany). The integrity of RNA preparations was assessed by electrophoresis on agarose gel. Total RNA (2 µg) was reverse-transcribed into cDNA by the random hexanucleotide technique using 100 U Moloney murine leukemia virus RT (RNaseH-; Invitrogen, Italy) and random primers p(dN)6 (Roche, Indianapolis, IN), according to the manufacturer. An aliquot of RT reaction (3 µl) was PCR-amplified in a final volume of 50 µl by using 200 µM concentration of dATP, dGTP, and dTTP and 10 µM dCTP, 0.5 U Taq–DNA polymerase (Amersham Pharmacia Biotech, Little Chalfont, UK), 0.2 µCi ({alpha}-32P)dCTP (Amersham Pharmacia Biotech; 3000 Ci/mmol), and 20 pmol each primer designed to amplify the NPR or the ß-actin mRNA coding sequences. For the human NPR-A mRNA amplification (ID: X15357), the following primes were used: upstream 5'-CGG TGG ACC ACC TGG AGT TCG-3', downstream 5'-GAG CAG GAG CCC GTC GTG GAA-3'; for the human NPR-B mRNA (ID: AJ005282): upstream 5'-GGC TAA GAA TGA CCA TTA TCG-3', downstream 5'-TAG CAG GAT CCC ATC ATA GAA-3'; and for the human NPR-C mRNA (ID: X52282): upstream 5'-GCA CAA GGA CTC TGA GTA CTC, downstream 5'-GAG GAG GAT GGC ATC GTG GAA-3'. The NPR–PCR products were expected to have the following sizes: NPR-A, 437 bp; NPR-B, 639 bp; and NPR-C, 606 bp. All transcripts were amplified as follows: 94°C for 4 min, 5 cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 2 min, 30 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min, followed by a final extension at 72°C for 10 min. RNA integrity and cDNA synthesis were verified by amplifying human ß-actin cDNA (ID: NM001101) with the following oligonucleotides: upstream 5'-GCA CTC TTC CAG CCT TCC-3', downstream 5'-GCG CTC AGG AGG AGG AAT-3' (fragment size, 193 bp). The primers for each mRNA were chosen in different exons to distinguish RNA products from possible contaminating DNA. The amplified fragments were separated on 5% polyacrylamide gel, and the radioactive gels were exposed and visualized by a PhosphorImager (MolecularDynamics, Sunnyvale, CA).

Analysis of 2',7'-dichlorofluorescein (DCF) fluorescence
DCF diacetate (DA) was dissolved in DMSO (Me2SO) at a final concentration of 1 mM and kept at -20°C in the dark; this solution was freshly diluted (10 µM/106 cells) before each experiment. The cells were loaded with the fluorescent indicator DCF–DA for 60 min at 37°C in the dark. DCF–DA diffuses through the cell membrane readily and is hydrolyzed by intracellular esterase to nonfluorescent DCF deacetylated, which is then rapidly oxidized to highly fluorescent DCF in the presence of ROS. The DCF fluorescence intensity is proportional to the amount of ROS formed intracellularly [30 ]. After the incubation with the fluorescent dye, the cells were washed two times, centrifuged at 700 g for 10 min, and resuspended in Na+ buffer. Fluorescence was measured under continuous magnetic stirring at 37°C in a Perkin–Elmer luminescence spectrometer Model LS-5 equipped with a chart recorder (Model R 100A), with an excitation wavelength at 485 nm and an emission wavelength at 530 nm, using 5 and 10 nm slits, respectively, for the two light paths. In the dose-response experiments, the cells were treated with different concentrations (10-7–10-11 M) of ANP for 6 h. In the experiments with calphostin-c and diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, the cells were pretreated for 1 h, and both substances were used at a final concentration of 10-7 M.

Reagents
RPMI 1640, trypsin 2.5%, glutamine, penicillin (100 UI/ml), and streptomycin (100 µg/ml) were from Eurobio Laboratoires (Le Ulis Cedex, France). FCS was from Gibco (Grand Island, NY). [3H]-Myristic acid (spec. act. 53 Ci/mmol) was from Amersham International (Bucks, UK). ANP (99–126), cANF (4–23), 8-Br-cGMP, calphostin-c, EIPA, trypan blue, DPI, 2-[N-morpholino]ethanesulfonic acid, Tris-HCl, and nigericin were from Sigma Chemical Co. (St. Louis, MO). DCF–DA, and BCECF/AM were from Molecular Probes (Eugene, OR). Ro-31-8220 and D609 were from Calbiochem (La Jolla, CA). Moloney murine leukaemia virus RT, random esaprimers, and NPR primers were from Invitrogen. Taq polymerase was from Amersham Pharmacia Biotech. Plates for thin-layer chromatography and all other chemicals of purest available grade were obtained from Merck (Darmstadt, Germany).

Statistical method
All data are presented as means ± SD. Statistical analysis was performed using the Student’s t-test.


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RESULTS
 
Intracellular acidification in human macrophages and THP-1 monocytes
To investigate whether ANP could induce pHi decrease in human macrophages and THP-1 monocytes, a wide concentration range (10-11–10-7 M) of ANP was used. The dose response of the ANP effect on pHi in macrophages and THP-1 monocytes is reported in Figure 1 . In this regard, the mean average pHi at rest was 7.14 ± 0.07, and low ANP concentrations (10-10–10-9 M) significantly decrease pHi, which shifted from basal value by 0.14 ± 0.05 pH units. In contrast, the average pHi at rest in THP-1 cells was 7.28 ± 0.04, and no significant ANP effects were observed (Fig. 1) .



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  		Figure 1. ANP effect on pHi in human macrophages and THP-1 monocytes. Human macrophages (M{Phi}; {blacksquare}) and THP-1 monocytes (•), grown in RPMI-1640, supplemented with 5% FCS, lowered to 0.5% before each experiment, were treated with ANP between 1011 and 10-7 M for 6 h, respectively, and were used for pHi determination, as reported in Materials and Methods. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. C, untreated cells.

A similar pHi decrease was observed when human macrophages and THP-1 monocytes were treated with EIPA, an inhibitor of the Na+/H+ antiport, within the micromolar concentration range, and pHi behavior is reported in Figure 2 . Our results show a significant decrease of pHi following 30 µM EIPA treatment, and the maximal effect was observed with 50 µM EIPA for both cell types.



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  		Figure 2. EIPA effect on pHi in human macrophages and THP-1 monocytes. Human macrophages (M{Phi}) ({blacksquare}) and THP-1 monocytes (•), grown in RPMI-1640, supplemented with 5% FCS, lowered to 0.5% before each experiment, were treated with EIPA, 10–50 µM, for 6 h, respectively, and were used for pHi determination, as reported in Materials and Methods. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. C, untreated cells.

It is interesting that the pHi decrease observed after 30 µM EIPA treatment in both cell types was comparable with that observed with 10-10 M ANP on human macrophages.

In Figure 3 , we report the time-course of pHi in the presence of 10-10 M ANP in human macrophages and 30 µM EIPA in THP-1 monocytes. The effect of ANP and EIPA was significant after 4 h of treatment and reached a maximal intracellular acidification after 6 h of treatment, and no significant differences were present between 6 and 12 h of treatment. For this reason, in experiments to be reported, pHi was modified using 10-10 M ANP for macrophages or 30 µM EIPA for THP-1 monocytes for 6 h.



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  		Figure 3. Time-course of intracellular acidification in human macrophages and THP-1 monocytes. Human macrophages (M{Phi}) ({blacksquare}) and THP-1 monocytes (•), grown in RPMI-1640, supplemented with 5% FCS, lowered to 0.5% before each experiment, were treated with 10-10 M ANP or with 30 µM EIPA, respectively, for different experimental times and were analyzed for pHi, as reported in Materials and Methods. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. C, untreated cells.

The modification of intracellular acidification induced by ANP treatment does not cause macrophage injury assessed by the trypan blue exclusion. In fact, cells were still 98–100% viable after 6 h of treatment (not shown). Conversely, we observed a significant mortality in THP-1 cells treated with EIPA: The percentage of cell death with 50 µM EIPA was ~50%, but it was reduced to 18% when the cells were treated with 30 µM EIPA for 6 h (not shown); similar results were also obtained for human macrophages.

As we have observed a different sensitivity to ANP exposure between human macrophages and THP-1 monocytes as far as pHi was concerned (see Fig. 1 ), the following experiments were performed only on human macrophages. To estimate whether the ANP-induced pHi decrease on human macrophages was dependent on the Na+/H+ antiport, we studied the pHi behavior in a Na+ buffer and in a choline buffer (Fig. 4 ) to keep quiescent the Na+/H+ antiport in the presence of 10-10 M ANP, 30 µM EIPA, and ANP plus EIPA. Our results indicated that in Na+ buffer, ANP has the same acidifing effect as EIPA, and ANP plus EIPA do not have an additive effect; in choline buffer, ANP or EIPA did all and singular not affect pHi (basal values, 7.07±0.03). These results indicate that the ANP effect on the intracellular acidification of human macrophages involves the inhibition of the Na+/H+ antiport.



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  		Figure 4. Effect of ANP and EIPA on pHi in human macrophages. Human macrophages (M{Phi}), grown in RPMI-1640, supplemented with 5% FCS, lowered to 0.5% before each experiment, were treated with 10-10 M ANP, 30 µM EIPA, or ANP plus EIPA for 6 h in Na+ buffer ({square}) and in choline buffer ({blacksquare}) and were analyzed for pHi, as reported in Materials and Methods. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. C, untreated cells.

Lipid messenger production after intracellular acidification
The role of DAG and PA, as second messengers and mitogens, has recently attracted considerable interest [31 ]. Changes in pHi observed in response to an array of ligands may be considered signaling events; in this regard, Gewirtz et al. [17 ] showed an enhancement of neutrophil degranulation and PLD activity when the Na+/H+ antiport is blocked. For this reason, we investigate a possible relationship between pH modulation and PLD activity.

PLD activity was studied after ANP treatment of human macrophages, and the involvement of PLD in PA production was investigated monitoring the transphosphatidylation reaction, which catalyzes the PE formation in the presence of ethanol. As the PA produced is rapidly metabolized and could arise from other sources, we have measured the formation of PE that is widely accepted as a direct measure of PLD activity [27 ]. The effect of ANP on PE production in human macrophages is reported in Figure 5 and suggests the following: ANP (10-10 M) induces a significant increase of PE production with respect to untreated cells; and 10-7 M calphostin-c, a potent inhibitor of PLD [32 ], has no effect on basal PLD activity nor does it affect cell viability throughout the experimental period (not shown), and calphostin-c pretreatment is able to inhibit the ANP effect totally. These results suggest an involvement of PLD in ANP-induced PA production.



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  		Figure 5. Effect on ANP on [3H]-myristate incorporation into PE in human macrophages. Human macrophages (M{Phi}) were labeled with [3H]-myristic acid (1 µCi) for 3 h at 37°C before the addition of 10-10 M ANP. Calphostin-c (calph; 10-7 M) was added 1 h before ANP treatment, and then the PE production was assessed, as reported in Materials and Methods. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. C, untreated cells; DPM, disintegration per minute.

As PA can be further metabolized into a bioactive lipid, such as DAG, the ANP effect on DAG intracellular production in human macrophages was investigated. In this regard (Fig. 6 ), we observed that 10-10 M ANP induces a significant increase of DAG with respect to untreated cells; 10-7 M calphostin-c, alone, has no effect, and calphostin-c pretreatment is not able to inhibit the ANP effect significantly; and 5 µM D609, specific inhibitor of PC–PLC, alone, is ineffective, and it induces a total inhibition with respect to ANP treatment. In the same experiments performed in choline buffer, the hormone effect on PA as well as on DAG levels was not evident (not shown).



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  		Figure 6. Effect of ANP on [3H]-myristate incorporation into DAG in human macrophages. Human macrophages (M{Phi}) were labeled with [3H]-myristic acid (1 µCi) for 3 h at 37°C before the addition of 10-10 M ANP, as reported above. Calphostin c (calph; 10-7 M) or 5 µM D609 was added 1 h before ANP treatment, and DAG production was assessed, as reported in Materials and Methods. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. C, untreated cells; DPM, disintegration per minute.

These data suggest that DAG accumulation was not directly dependent on PA production but showed a possible relationship between ANP-induced intracellular acidification and PL activities.

ANP and EIPA effect on ROS production
The possible effect of ANP on ROS production was investigated over a wide concentration range (10-7–10-11 M). Human macrophages were labeled with DCF–DA and stimulated with ANP for 6 h as reported in Materials and Methods, and ROS production was evaluated. Figure 7 shows an ANP dose-dependent increase of ROS production, which was always above basal levels with a maximum at 10-10 M ANP and proved to be specific for macrophages (see inset).



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  		Figure 7. Dose-response of ANP on ROS production in human macrophages. Human macrophages (M{Phi}) were labeled with DCF–DA, and ROS production was evaluated after 6 h of treatment with different concentrations (10-7–10-11 M) of ANP, as reported in Materials and Methods. Results are expressed as fluorescence intensity, reported as fluorescence units (F.U.) of cells loaded with only DCF–DA (C) or also treated with 300 µM H2O2 for 1 h as a positive control. Data represent the mean ± SD values for four experiments. (Inset) The same experiments performed on THP-1 monocytes. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells.

PA and DAG have been correlated with the activation of NADPH oxidase and ROS production [33 ]: In particular, PA should act directly, and DAG action is mediated by PKC. The possible relationship between ANP treatment and ROS production was investigated, treating human macrophages with calphostin-c, Ro-31-8220, an inhibitor of PKC, and DPI, a specific inhibitor of NADPH oxidase. In Figure 8 , it is observed that ANP induced a significant increase of ROS production with respect to untreated cells; the calphostin-c or Ro-31-8220 alone does not affect ROS production, and the pretreatment with the inhibitors is able to inhibit the ANP-induced ROS production only partially; and 10-7 M DPI alone is ineffective, and it induces a total inhibition with respect to ANP effect. These results suggest that ANP could stimulate ROS production through the involvement of NADPH oxidase. To estimate whether the ANP-induced ROS production in human macrophages was dependent on the Na+/H+ antiport, we studied the ROS production in the presence of 30 µM EIPA (Fig. 8 , inset): Our results show that the pattern of EIPA-induced ROS production is definitely superimposable to that reported for ANP treatment, suggesting a possible link between ANP-induced intracellular acidification and ROS production in human macrophages.



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  		Figure 8. ROS production in human macrophages in the presence of ANP. Human macrophages (M{Phi}) were labeled with DCF–DA (C), and ROS production was evaluated after 6 h of specific treatment with 10-10 M ANP, as reported in Materials and Methods. Calphostin c (calph; 10-7 M), 10-7 M DPI, and 5 µM Ro-31-8220 were added 1 h before ANP treatment. Results are expressed as fluorescence units (F.U.), as reported in Figure 7 . Data represent the mean ± SD values for four experiments. As indicated in the inset, 30 µM EIPA, alone or plus inhibitors, was added, and ROS production was estimated. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells.

NPR-A, NPR-B, and NPR-C mRNAs are present in human THP-1 monocytes and macrophages
To verify whether the different behavior between the two cell types, with respect to ANP stimulation, is a result of different expression of NPRs, we performed radioactive RT-PCR on total RNA from human macrophages and THP-1 monocytes. Figure 9 shows that all three mRNAs are represented in both cell types. Even if an absolute quantification of the distinct NPR mRNAs is not possible because the PCR reaction was performed under saturated conditions (35 cycles), we can clearly conclude that a lower level of NPR-B mRNA expression is observed in macrophages as compared with THP-1 monocytes (Fig. 9B , compare lanes 2 and 1). ß-Actin mRNA does not show any change in the expression level in both cell types (Fig. 9D , compare lanes 1 and 2).



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  		Figure 9. Radioactive RT-PCR of NPR-A, NPR-B, and NPR-C mRNAs from human THP-1 monocytes and macrophages. The same RT reaction was equally divided and used to amplify NPR-A mRNA (A, lanes 1 and 2), NPR-B mRNA (B, lanes 1 and 2), and NPR-C mRNA (C, lanes 1 and 2) in THP-1 monocytes and human macrophages (M{Phi}). The products were separated on a polyacrylamide gel and acquired by a phospho-imager. ß-Actin mRNA was also detected in both cell types (D, lanes 1 and 2). One-half of the PCR product was loaded for samples A, B, and C and one-tenth for sample D.

Selectivity and functional activity of ANP receptors on human macrophages
To determine which NPR mediates the inhibitory effect of ANP on pHi in human macrophages, experiments to be reported in the following were performed with 8-Br-cGMP, an analog not hydrolyzable of cGMP and with cANF (4–23), the ring-deleted analog of ANP specific for C-receptor in a wide range of concentrations (10-7 M–10-11 M). Figure 10 shows that 1 mM 8-Br-cGMP was ineffective on pHi, and 10-10 M cANF concentration was found to be the most effective and was able to mimic the ANP-induced pHi decrease. These results suggest an involvement of C-receptor in the ANP effect on pHi decreases in human macrophages. To investigate whether the activation of specific PLs was related to NPR-C [2 ], we studied the cANF effect on PLD and PC–PLC monitoring PE and DAG production, respectively. In this regard (Fig. 11a and 11b ), we observed that the specific NPR-C ligand cANF (1010 M) was able to mimic ANP effect on PE as well as DAG production (see Figs. 5 and 6 for comparison). Moreover, to determine the NPR-C involvement in ROS production, we treated human macrophages with 10-10 M cANF, observing that as already reported above for ANP, there was a significant ROS production with respect to untreated cells, where the pretreatment with specific inhibitors, calphostin-c or Ro-31-8220, inhibited the cANF effect only partially, and the ROS production was totally inhibited by DPI pretreatment (Fig. 11c) .



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  		Figure 10. Effect of 8-Br-cGMP and cANF on pHi in human macrophages. Human macrophages (M{Phi}), grown in RPMI-1640, supplemented with 5% FCS, lowered to 0.5% before each experiment, were treated with 10-10 M ANP, 1 mM 8-Br-cGMP, or a wide concentration range of cANF (10-7–10-11 M) and were analyzed for pHi, as reported in Materials and Methods. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. C, untreated cells.



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  		Figure 11. cANF effect on PE, DAG, and ROS production in human macrophages. Human macrophages (M{Phi}) were treated for PE (a), DAG (b), and ROS (c) production as reported in the legends of Figures 7 8 and 10 , respectively, before 10-10 M cANF addition. Data are reported as mean ± SD of four different experiments. *, P < 0.05, as from a Student’s t-test, with respect to untreated cells. calph, Calphostin c; C, untreated cells; DPM, disintegration per minute.


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DISCUSSION
 
Recently, Vollmar et al. [15 ] showed that ANP at physiological concentrations influences two important functions of macrophages, i.e., phagocytosis and oxidative burst activity, but the mechanism involved is not known. Our results suggest that changes of cytoplasmic pH, exhibited by human macrophages in response to ANP, play a role in regulating ROS production. The effects were found at concentrations as low as 10-10 M, levels that fall in the range of ANP plasmatic concentrations, and the decreased response to higher concentrations of ANP may be explained by receptor down-regulation after the ANP treatment [34 ]. The mechanism through which ANP induced pHi decrease is yet unknown, but some reports propose that ANP could inhibit Na+/H+ antiport through an increase of intracellular Ca2+ concentrations [35 ].

Gewirtz et al. [17 ] showed that neutrophil degranulation and PLD activation are enhanced if the Na+/H+ antiport is blocked by amiloride or by its analogs such as EIPA. The mechanism by which EIPA acidification increases PLD activity is yet unclear. It is conceivable that a slightly acid pH directly enhances PLD activity, as supported by the observation that isolated PLDs have a maximal activity at acidic pH [17 ]. Considering that amiloride interferes with chemotaxis presumably through the polymerization of actin [36 ] and enhances PLD activity, the pH effect on PLD might be mediated through cytoskeletal rearrangements. It is known that the activation of PLD also occurs in response to a wide array of hormones and growth factors [37 ] and that PA, the initial product of PLD, acts as an intracellular signaling effector. In this regard, our observations indicate that ANP gives rise to an increase of intracellular PA concentrations, in parallel with the hormone-induced pHi decrease. PA and DAG are involved in many cellular responses, such as PKC isoform stimulations [37 , 38 ] and/or mitogen-activated protein kinase signal transduction [39 ]. In particular, PA changes in stimulated cells are generally of a similar magnitude as DAG changes but are more sustained and able to modulate long-term events such as secretion, contraction, and proliferation [40 ]. It is interesting that our results show that ANP also induces intracellular DAG accumulation in human macrophages, independently of PA, but depending on a PC–PLC involvement. DAG and PA have been proposed as intracellular messengers during the activation of the repiratory burst [41 ], and it is known [33 ] that PA alone or together with DAG induces ROS production through NADPH oxidase activation, also if the mechanism of receptor-mediated activation is not completely understood. Our experiments on ANP-induced ROS production in human macrophages confirm the involvement of DAG and PA and suggest that NADPH oxidase activation is dependent on lipid mediators through PLD and PC–PLC involvement. Such a mechanism of ANP-induced ROS production in human macrophages is conceivable, as calphostin-c, inhibitor of PLD, and Ro-31-8220, inhibitor of DAG-dependent PKC, were able to inhibit the effect of hormone only partially, which was conversely, totally inhibited by DPI, a specific inhibitor of NADPH oxidase. NADPH oxidase is responsible for converting oxygen to toxic products (O·2), and its normal function is vital for resistence to bacterial infections [42 ], as suggested by chronic granulomatous disease syndrome, in which various defects of the protein components of the NADPH oxidase result in a loss of the respiratory burst and in the inability to resist to many infections [43 ]. It is interesting that our data suggest a specific role of ANP-induced intracellular acidification for NADPH oxidase activation and ROS production. The intracellular acidification in human macrophages is induced following exposure to physiological concentrations of ANP, and the additive action of DAG and PA on NADPH oxidase could be responsible for ROS production.

Finally, our data demonstrate for the first time that human macrophages as well as THP-1 monocytes express mRNA coding for NPR-A, NPR-B, and NPR-C, but the mechanism involved seems specific for macrophages, as the THP-1 monocytes, in spite of expression of all NPRs, are insensitive to ANP, and in these cells, the intracellular acidification was induced only by pharmacological treatment with EIPA. The insensitivity of THP-1 cells to ANP might be explained by the inability of NPRs to mediate such effects. However, these results are in good agreement with a recent observation, indicating that NPs are produced in THP-1 monocytes after phorbol-ester treatment leading to differentiation into macrophages [7 ].

Several evidences support a role for NPR-C in ANP-induced biological effects [44 ], and our results show that cANF, the ring-deleted analog of ANP, which is specific for NPR-C, is able to mimic ANP-induced effects on pHi, PL activities, and ROS production in human macrophages, suggesting a NPR-C involvement, in good agreement with recent observations [45 ] that such a receptor is involved in ANP-induced inhibition of cyclooxygenase-2 in mouse marrow macrophages.

In conclusion, we may suggest new, additional, physiological roles for ANP: Its specific effect on macrophages could be relevant for the modulation of inflammatory response and contribute to local and systemic aspects of host-defense mechanisms.

Received July 29, 2002; revised December 2, 2002; accepted January 2, 2003.


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