1 Institut National de la
Santé et de la Recherche Médicale Unité 64, Because atrial
natriuretic peptide (ANP) is considered to play a role in lung
physiology and pathology, our aim was to characterize natriuretic
peptide receptors in cultured rat alveolar type II (ATII) cells.
Guanylate cyclase A- and B-receptor but not clearance-receptor mRNAs
were detected by reverse transcription-polymerase chain reaction. The
absence of clearance-receptor expression in ATII cells was confirmed by
competitive inhibition of ANP binding; ANP (0.1-100 nM) decreased
the binding of 125I-ANP, whereas
C-ANP-(4
atrial natriuretic peptide; guanosine 3',5'-cyclic
monophosphate; cyclic nucleotide export; amiloride-sensitive sodium-22
influx
ATRIAL NATRIURETIC PEPTIDE (ANP), which belongs to the
family of natriuretic peptides, plays a major role in electrolyte and volume homeostasis through potent biological effects including natriuresis, diuresis, and vasorelaxation. Originally discovered in rat
atria, ANP is an ubiquitous hormone, with its gene being expressed in
several tissues including the lungs (11). The lung, besides being a
target organ for ANP from atrial origin, is also a site of synthesis
and release of bioactive ANP (12). The actions of ANP are receptor
mediated. There are at least two major biochemical and functional
distinct classes of ANP receptors, guanylate cyclase (GC) and clearance
receptors. GC receptors mediate the functional effects of the
natriuretic peptides, whereas clearance receptors, which do not possess
a GC moiety, are implied in the intracellular degradation of these
peptides (21). Two subtypes of GC receptors have been described: GC-A
receptors with a high affinity for ANP and brain natriuretic peptide
and GC-B receptors that are specific for C-type natriuretic peptide
(CNP) (21). The lung has the highest tissue concentration of specific
ANP binding sites. Using cytochemical studies, Rambotti and Spreca (30)
localized ANP-stimulated GC activity in rat alveolar epithelial cells,
but until now, natriuretic peptide receptor subtypes in alveolar
epithelial type II (ATII) cells have not been yet characterized.
The functional role of lung ANP is not well known. A previous report
(15) has shown that, in guinea pigs, ANP administration had protective effects in cardiogenic and noncardiogenic lung edema;
the mechanisms for the protective effects were not elucidated. On the
other hand, a recent study (28) performed in isolated perfused
liquid-filled rat lungs reported that ANP inhibited active Na transport
and increased alveolar epithelial permeability, thus decreasing lung
edema clearance. In alveolar epithelium, ATII cells maintain the
alveolar space in the dry state by an active transport of fluid and
electrolytes, primarily Na, from the air space to the interstitium
(22). Na enters the apical membrane of ATII cells mainly through
amiloride-sensitive Na channels and is extruded at the basolateral side
of the cells by ouabain-sensitive Na-K-adenosinetriphosphatase
(ATPase). Presently, whether ANP may regulate active Na transport in
ATII cells is unknown.
Our goal was to characterize the natriuretic peptide receptor subtypes
and to evaluate the effect of ANP on Na transport in ATII cells that
had been isolated from rat lungs. Our results demonstrate that cultured
ATII cells express both GC receptor subtypes, with a marked functional
predominance of GC-A over the GC-B receptor, and do not possess
clearance receptors. In addition, ANP decreases the amiloride-sensitive
22Na influx in these cells, which
suggests that in vivo ANP tends to inhibit Na reabsorption. Therefore,
the preventive role of ANP on pulmonary edema does not seem to be
explained by its direct effects on Na transport in ATII
cells.
Cell Culture
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
23), a specific ligand of clearance receptors, was
ineffective. ANP induced a dose-dependent increase in guanosine 3',5'-cyclic monophosphate (cGMP) production, with a
threshold of 0.1 nM, whereas the response to C-type natriuretic peptide was weak and was observed only at high concentrations (100 nM). In ATII
cells cultured on filters, 1) ANP
receptors were present on both the apical and basolateral surfaces and
2) cGMP egression was polarized, as
indicated by the greater ANP-induced cGMP accumulation in the
basolateral medium, and was partially inhibited by probenecid, an
organic acid transport inhibitor. Influx studies demonstrated that ANP
decreased the amiloride-sensitive component of
22Na influx but did not change
ouabain-sensitive 86Rb influx. In
conclusion, ATII cells behave as a target for ANP. ANP activation of
guanylate cyclase A receptors produces cGMP, which is preferentially
extruded on the basolateral side of the cells and inhibits the
amiloride-sensitive Na-channel activity.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
ATII cells were isolated from pathogen-free male Sprague-Dawley rats
(200-250 g) as previously described (7). Briefly, pooled cells
from three rats/experiment were prepared as follows. The rats were
injected intraperitoneally with 30 mg/kg of pentobarbital sodium and
intravenously with 1 U/g of sodium heparinate. After a tracheotomy was
performed, the animal was exsanguinated. Solution I (40-50 ml), which contained (in mM) 140 NaCl, 5 KCl, 2.5 Na phosphate buffer, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2 CaCl2, and 1.3 MgSO4, pH 7.4, at 22°C, was
perfused through the air-filled lungs by way of the pulmonary artery to clear the vascular space of blood. The lungs were removed from the
thorax and washed to total lung capacity (8-10 ml) five times with
solution II, which contained (in mM)
140 NaCl, 5 KCl, 2.5 Na-phosphate buffer, 10 HEPES, 6 D-glucose, and 0.2 ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, to remove macrophages and two times with
solution I. Then the lungs were filled
with 12-15 ml of 40 U/ml elastase from porcine pancreas
(Worthington Biochemical, Freehold, NJ), crystallized two times,
prepared in solution I, and incubated
in a shaking water bath for 10 min at 37°C in air atmosphere.
Additional elastase solution was then instilled for another 10-min
incubation. The lungs were minced in the presence of deoxyribonuclease
I, and 5 ml of fetal calf serum (FCS) were added to stop the effect of
elastase. The lungs were then sequentially filtered through 150- and
30-mm nylon mesh. The filtrate was centrifuged at 130 g for 8 min. The cell pellet was
suspended in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM D-glucose at 37°C. The
cell suspension was plated at a density of
106
cells/cm2 in
25-cm2 bacteriological plastic
dishes to aid in the removal of macrophages by differential adherence.
After incubation at 37°C in a 5%
CO2 incubator for 1 h, the
unattached cells in the suspension were removed and centrifuged at 130 g for 8 min. The resulting cell pellet
(70% purity, >95% viability, 8-10 × 106 cells/rat) was plated at a
density of 7-10 × 105
cells/cm2 in 6-, 12-, or 24-well
culture plates. The culture medium consisted of DMEM containing 25 mM
D-glucose, 10 mM HEPES, 23.8 mM
NaHCO3, 2 mM
L-glutamine, 10% FCS, 50 U/ml
of penicillin, 50 mg/ml of streptomycin, and 10 mg/ml of gentamicin.
Cells were incubated in a 5%
CO2-95% air atmosphere. After 24 h, the culture medium was changed to a serum-free defined medium
composed of DMEM supplemented with 2 mM
L-glutamine, 50 U/ml of
penicillin, 50 µg/ml of streptomycin, 10 µg/ml of gentamicin, 5 µg/ml of insulin, and 10 µg/ml of transferrin. The cells were
cultured for an additional 24-h period. The cell purity after 24 h was
90 ± 2% as assessed by a characteristic fluorescence with
phosphine 3 R as previously described (7). Contaminating cells were
essentially macrophages.
A series of experiments was performed on the cells grown in Transwell chambers to obtain polarized cultures. Cells were cultured for 3 days in DMEM containing 10% FCS until they formed a continuous monolayer with tight junctions. Then the medium was changed to the defined medium described above, and the cells were cultured for an additional 24-h period.
Cultured rat mesangial cells used as control cells for the expression of natriuretic peptide-receptor mRNA by rat ATII cells were obtained as previously described (8).
Expression of Natriuretic Peptide-Receptor mRNA
Reverse transcription. Total RNA was extracted from cultured rat ATII cells and rat mesangial cells with Trizol reagents (Life Technologies, Cergy-Pontoise, France) according to a method derived from that of Chomczynski and Sacchi (5). Two micrograms of total RNA were reverse transcribed with 10 mM dithiothreitol, 1 mM deoxynucleotide mixture, 4 U/µl of Moloney murine leukemia virus (GIBCO BRL) reverse transcriptase (RT), and RT buffer [75 mM KCl, 3 mM MgCl2, and 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.3]. The total volume was 20 µl. After 2 h of incubation at 37°C, the reaction was stopped by placing the tubes on ice for 10 min.Polymerase chain reaction. Polymerase chain reaction (PCR) was performed with Taq DNA polymerase (Promega) with specific GC-A-, GC-B-, and clearance-receptor primers. The primers were derived from the rat cDNA sequence of the corresponding rat GC receptors and from the bovine cDNA sequence of clearance receptors according to Nunez et al. (26).
Primers for the GC-A receptor were sense 5'-GGGGATGTGGAAATGAAGGGC-3' ( primer 1) and antisense 5'-TCACAGGAGAAGCAAGGGTAACCGGC-3' ( primer 2), corresponding to bases 3321-3341 and 3868-3893, respectively (33).
Primers for the GC-B receptor were sense 5'-GATGAACCTCATTGCGGG-3' ( primer 1) and antisense 5'-GAGTTCAAACAGAACTTGCCG-3' ( primer 2), corresponding to bases 1059-1076 and 1756-1776 (20).
Primers for the clearance receptor were sense 5'-ATCGTGCGCCACATCCAGGCCAGT-3' ( primer 1) and antisense 5'-TCCAAAGTAATCACCAATAACCTCCTGGGTACCCGC-3' ( primer 2) (26).
The amplification reaction mixture consisted of 1× PCR buffer (50 mM KCl, 0.1% Triton X-100, and 10 mM Tris · HCl, pH 9), 2 mM MgCl2, 0.5 mM deoxynucleotide mix, 1 µM each primer, and 500 U/ml of Taq DNA polymerase. The total volume was 50 µl. A Kontron Instruments Trio Thermoblock thermocycler was used with the following conditions: 5-min initial denaturation at 94°C; 35 cycles of 40-s denaturation at 94°C, 1-min annealing at 55°C, and 2-min extension at 72°C; and 7-min final extension at 72°C. The products of expected sizes were identified by agarose gel (2%) electrophoresis. For negative controls, amplification of the receptors was attempted on a sample in which the RT enzyme had been omitted in the RT reaction. After electrophoresis and ethidium bromide staining, DNA bands were visualized with an ultraviolet transilluminator.
Restriction Digestion on PCR Products of ATII Cells
In some experiments, the PCR products were removed and purified from the agarose gel with a Geneclean II kit (Bio 101, La Jolla, CA). Eight microliters of the purified PCR products of the GC-A and GC-B receptors were incubated for 75 min at 37°C with each of the following enzymes: GC-A receptor, 10 units Pst I in H buffer (10 mM MgCl2, 100 mM NaCl, 1 mM dithioerythritol, and 50 mM Tris · HCl, pH 7.5); and GC-B receptor, 10 units BamH I in B buffer (5 mM MgCl2, 100 mM NaCl, 1 mM 2-mercaptoethanol, and 10 mM Tris · HCl, pH 8). The products of this restriction digestion were identified by agarose gel according to the method mentioned above.Guanosine 3',5'-Cyclic Monophosphate Production Determination
In a first set of experiments, guanosine 3',5'-cyclic monophosphate (cGMP) production was measured in the presence of increasing concentrations of ANP and CNP (Peninsula Laboratories, Merseyside, UK) on cells grown in 12-well dishes over 2 days and arriving at confluence according to the method previously described (2). Briefly, cells were rinsed and preincubated for 15 min with 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma Chemical, St. Louis, MO). The medium was discarded, and ANP or CNP was added to the culture medium for 5 min at 37°C in room atmosphere in the presence of 1 mM IBMX. The supernatants were then collected, and 1 ml of 0.1 M HCl was added to each well for extraction of intracellular cGMP. After 5 min at 4°C, these extracts were pooled with their respective incubation media. The total cGMP content was determined by radioimmunoassay after acetylation of the samples and standards with 125I-cGMP (Radiochemical Centre, Amersham) as a tracer and an anti-cGMP antibody (Pasteur Institute, Marnes la Coquette, France).A second set of experiments was performed in the presence of ANP to
determine whether cGMP release was polarized, according to the method
previously described (1). In addition, these experiments allowed us to
compare the efficiency of ANP on cGMP production when added to the
basolateral or apical chamber. Cells were grown on 0.4-µm Costar
filters in 12-well dishes and studied 4 days later. For technical
reasons, the incubation period of ANP was longer with cells grown on
filters than with those grown on the bottom of the wells. First, to
quantify cGMP leakage through the cell layer, the cells were incubated
for 1 h with 125I-cGMP (1,000 counts/min) alternately in the apical and basolateral baths. At the end
of this incubation, the cells were loaded with 1 mM IBMX for 5 min.
Culture media were collected separately, and radioactivity was measured
in both compartments. 125I-cGMP
diffusion from the apical to the basolateral chamber and vice versa did
not differ and represented 8 ± 1% of the total radioactivity
(n = 30 cells). The cells
were then rinsed and incubated with culture medium supplemented with
0.1 mM IBMX (500 µl in each chamber) in a 5%
CO2-95% air atmosphere at
37°C. ANP (0.1 µM) was added alternately to the apical and
basolateral chambers or simultaneously to both compartments (ANP
leakage from one bath to another, determined in preliminary studies by
diffusion of 125I-ANP, was
<2%). In some experiments when ANP was present in both compartments,
probenecid (0.3 mM), a competitive antagonist of organic acid
transport, was added simultaneously with the peptide. After 15 min of
incubation, 200 µl of the supernatant were collected in each chamber.
The remaining medium was collected 35 min later. Intracellular cGMP was
extracted by incubating the cells with 0.1 M HCl for 5 min at 4°C.
The protein content per well was determined by the Bradford (4) method
with bovine serum albumin (BSA) as a standard. cGMP concentrations were
measured in all the samples, and the total release in each compartment
at 1 h was calculated using the concentrations measured in the
corresponding samples collected after 15 and 60 min. The results are
expressed as picomoles of cGMP per milligram of protein. The
concentrations of cGMP found after 60 min in the apical and basolateral
media were corrected by taking into account the coefficient of leakage
according to the following formulas (1): a = [A 0.08(A + B)]/0.84 and b = [B
0.08(A + B)]/0.84, where A and B are the concentrations measured in the
apical and basolateral chambers, respectively, and a and b are the
corrected concentrations, respectively.
125I-ANP Binding on Cultured ATII Cells
The cells in 12-well dishes were rinsed twice and incubated in 0.4 ml of culture medium supplemented with 0.1% BSA. The cells were incubated at 4°C in room atmosphere in the presence of 100 pM 125I-ANP for different periods of time. At the end of the incubation period, the medium was discarded, and the cells were rinsed three times with 2 ml of ice-cold 0.16 M NaCl. Then they were solubilized in 0.4 ml of 1 M NaOH. The cell-associated 125I radioactivity was counted in a gamma counter (Micromedic). Specific binding was defined as the difference between total binding and nonspecific binding obtained in the presence of 1 µM unlabeled rat ANP. Preliminary studies of 125I-ANP binding as a function of time had determined that a plateau was reached within 90-180 min of incubation at 100 pM. Consequently, competitive-inhibition studies were performed by incubating 125I-ANP for 120 min in the presence or absence of unlabeled ANP or C-ANP-(422Na Influx Studies
The measurement of 22Na flux through amiloride-sensitive Na channels was performed as previously described (6). After removal of the culture medium, the cells in 12-well dishes were rinsed twice and preincubated for 20 min at 37°C in a buffered Na-free solution containing (in mM) 137 N-methylglucamine, 5.4 KCl, 1.2 MgSO4, 2.8 CaCl2, and 15 HEPES (pH 7.4) with from 1 to 100 nM ANP. At the end of the preincubation, the Na-free solution was replaced by the uptake solution containing (in mM) 14 NaCl, 35 KCl, 96 N-methylglucamine, and 20 HEPES (pH 7.4) with 1 mM ouabain and 0.5 µCi/ml 22NaCl (37 MBq/mg Na; Radiochemical Centre, Amersham) in the absence or presence of 10 µM amiloride. After a 5-min incubation period, uptake was stopped by washing the cells three times with 1 ml/well of an ice-cold solution containing (in mM) 120 N-methylglucamine and 20 HEPES (pH 7.4). Under these conditions, previous experiments indicated that total 22Na uptake increased with time for 20 min (6). The cells were solubilized in 0.5% Triton X-100. Tracer activities were determined by liquid scintillation counting, and the remaining volume of each sample was used for assessing the protein content per well. Amiloride-sensitive 22Na influx was calculated as the difference between uptake in the absence or presence of amiloride. Results are expressed in nanomoles per milligram of protein per 5 min.86Rb Influx Studies
The measurement of Rb influx was performed as previously described (7). Assays were performed at 37°C in buffered solution A of the following composition (in mM): 120 NaCl, 5 RbCl, 1 MgSO4, 0.15 Na2HPO4, 0.2 NaH2PO4, 4 NaHCO3, 1 CaCl2, 5 glucose, 2 lactate, 4 essential and nonessential amino acids, and 20 HEPES. The osmotic pressure of solution A was adjusted by the addition of mannitol to 350 mosM, and the pH was adjusted to 7.4. After removal of the culture medium, the cells in 24-well plates were washed two times and preincubated for 20 min in solution A containing 0.1 µM ANP. At the end of the incubation, the solution was replaced by the uptake solution: solution A supplemented with 1 µCi/ml 86RbCl (370 MBq/mg Rb) in the absence or presence of 1 mM ouabain. After a 5-min incubation, uptake was stopped by washing the cells three times with 0.5 ml/well of an ice-cold rinsing solution containing (in mM) 140 N-methylglucamine, 1.2 MgCl2, 3 BaCl2, 10 HEPES, and 0.1% BSA (pH 7.4). The cells were then solubilized in 0.5% Triton X-100, and tracer activities and protein content per well were determined as described in 22Na Influx Studies. Ouabain-sensitive Rb influx, reflecting Na-K-ATPase activity, was calculated as the difference between 86Rb influx measured in the absence or presence of 1 mM ouabain. Results are expressed in nanomoles per milligram of protein per 5 min.Statistical Methods
Results are given as means ± SE. The data were analyzed with Student's t-test for unpaired or paired values or one-way analysis of variance as appropriate. ![]() |
RESULTS |
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Natriuretic Peptide-Receptor mRNA Determination by RT-PCR
Figure 1 shows the amplification products after RT-PCR of GC-A-, GC-B-, and clearance-receptor mRNA from rat mesangial and ATII cells. The bands were of the predicted sizes, i.e., 573 base pairs (bp) for the GC-A and clearance receptors and 718 bp for the GC-B receptor (20, 26, 33). A band was observed for each type of receptor in the mesangial cells. In contrast, in the ATII cells, RT-PCR was positive for the GC-A and GC-B receptors but negative for the clearance receptor. These results were consistently found for both types of cells in three separate experiments. A restriction digestion analysis of the products obtained from amplification with ATII cells was then performed. The amplification products of GC-A receptors digested with Pst I and of GC-B receptors digested with BamH I gave, on the basis of their sequences, the expected products of 321 and 252 bp for the GC-A receptor and 574 and 144 bp for the GC-B receptor (20), respectively (Fig. 2).
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Effects of ANP and CNP on cGMP Production in ATII Cells
The physiological concentrations of ANP and CNP, which were close to the dissociation constant values of their respective receptors, were taken into account when the concentrations used to test the effects on cGMP production, ranging from 10 pM to 100 nM, were chosen. The stimulation of cGMP production by ANP was concentration dependent (Fig. 3). A significant increase occurred at a threshold of 0.1 nM, inducing a cGMP accumulation of 3.76 ± 0.64 (n = 9 cells) vs. 1.34 ± 0.19 pmol · mg protein
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When ATII cells were grown on filters, total cGMP production at 60 min was compared after exposure to 0.1 µM ANP added to the apical or basolateral chamber or simultaneously to both compartments. cGMP production was lower after basolateral stimulation (75 ± 27 pmol/mg; n = 7 cells) than after apical stimulation (411 ± 96 pmol/mg; P < 0.05; n = 7 cells). The cellular stimulations on both poles were additive as shown by the cGMP level measured after the addition of ANP (0.1 µM) simultaneously to both compartments (485 ± 24 pmol/mg; n = 7 cells).
The polarity of cGMP egression was evaluated after incubation with ANP for 15 and 60 min. cGMP release was higher in the basolateral than in the apical chamber (P < 0.01) regardless of the side of stimulation by ANP and the period of incubation (Fig. 4). When data at 60 min were corrected to take into account the leakage of cGMP, the difference in concentration between both chambers was accentuated [basolateral stimulation: 70 ± 39 (basolateral) vs. 4.6 ± 3.0 (apical) pmol/mg protein, P < 0.01; apical stimulation: 336 ± 129 (basolateral) vs. 74 ± 18 (apical) pmol/mg protein, P < 0.01]. Intracellular concentrations of cGMP (8 ± 1 pmol/mg after basolateral stimulation and 14 ± 4 pmol/mg after apical stimulation) were low compared with the concentrations in the extracellular basolateral compartment at 60 min.
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To test the role of organic anion transporters on cGMP egression, probenecid (0.3 mM) was added in both compartments simultaneously with ANP. Probenecid markedly attenuated the gradient between the basolateral and apical cGMP accumulations after 15 (26 ± 7 vs. 90 ± 9 pmol/mg without probenecid) or 60 min (54 ± 12 vs. 209 ± 24 pmol/mg; P < 0.01). Probenecid affected this gradient essentially by decreasing cGMP accumulation in the basolateral chamber (Fig. 5). Total cGMP production at 60 min was slightly decreased by probenecid (434 ± 19 vs. 485 ± 24 pmol/mg without probenecid; P < 0.05).
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125I-ANP Binding Studies in ATII Cells
To confirm the absence of expression of the clearance receptors in ATII cells, competitive binding studies of 125I-ANP were performed with unlabeled ANP and C-ANP-(4
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Effect of ANP on Na Transport in ATII Cells
ATII cells were incubated for 20 min with physiological concentrations of ANP from 1 to 100 nM. ANP (100 nM) significantly reduced the amiloride-sensitive 22Na uptake, but lower concentrations were ineffective (Fig. 7). In contrast, ANP (100 nM) did not modify Na-K-ATPase activity as estimated by the ouabain-sensitive 86Rb influx (141 ± 13.5 vs. 161 ± 19.5 nmol · mg protein
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DISCUSSION |
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It is established that the lungs are an extra-atrial source of ANP contributing to the circulating ANP pool (12). Several lines of evidence from in vivo studies support the view that the lungs, besides synthesizing ANP, possess functional ANP receptors. In anesthetized rats, a high labeling was observed in lungs, particularly in the alveolar epithelial cells, after infusion of 125I-ANP (3). In addition, there is morphological evidence that ANP activates particulate GC in rat ATII cells (30).
The present work was done to characterize the functional natriuretic peptide receptor subtypes in ATII cells. Our results unveil the presence of ANP receptors with GC activity and the absence of clearance receptors. The presence of functional GC-A receptors in alveolar cells was supported by the following evidence: 1) GC-A-receptor mRNA was detected by RT-PCR; 2) ANP markedly stimulated cGMP production in a concentration-dependent manner, with a low threshold concentration (100 pM); and 3) ANP concentrations inhibiting 125I-ANP binding were identical (100 pM to 0.1 µM) to those used to induce cGMP accumulation, which confirmed the relationship between the presence of ANP binding sites and the stimulation of GC activity.
Although GC-B-receptor mRNA was detected in ATII cells, CNP, which selectively activates the GC-B receptor (21), exhibited only a weak effect on these cells; cGMP production required a 1,000-fold greater concentration for CNP than for ANP to produce responses of similar magnitude, suggesting that GC-B receptors were expressed with a very low density at the surface of ATII cells. In a cortical collecting duct cell line, Millul et al. (23) detected GC-A and GC-B receptors by RT-PCR amplification. However, the threshold of membrane GC production was ~1,000 times higher with CNP than with ANP, and these authors failed to demonstrate specific binding of 125I-CNP on these cells. It cannot to be excluded that, in that study as well as from our own results, the decrease in GC-B activity after CNP is the result of experimental condition (i.e., cell culture in vitro). However, it must be emphazised that Terada et al. (34), in isolated segments of the nephron, observed the same discrepancy between GC-B-receptor mRNA detection and functional expression of the encoded protein. Taken together, our results suggest that ATII cells are a target for ANP but not for CNP.
Experiments performed on polarized cells demonstrated that cGMP production was induced when ANP was added on the apical or basolateral side of ATII cells. In the absence of a significant leakage of ANP from one chamber to the other, this result is in favor of the presence of GC-A receptors on both sides of cells. Because cGMP formation was greater when ANP was added on the apical side of cells, it may be hypothesized that GC-A-receptor density was greater on this side. However, this result could also be explained by a greater affinity of ANP for GC-A receptors expressed on the apical side or by its degradation on the basolateral side. Interestingly, in epithelial cells from the inner medullary collecting duct, such a polarized distribution of ANP receptors has been previously observed (31), but ANP receptors were present predominantly on the basolateral membrane. Extrapolation of our results to the in vivo situation suggests that ATII cells could be activated by ANP of vascular origin via the GC-A receptors present on their basolateral side and by ANP of local pulmonary origin that would bind the apical receptors.
Although functional studies generally focus on cGMP interactions with intracellular effectors [e.g., ion channels (27), phosphodiesterases (14), or protein kinases (17)], recent reports indicate that extracellular cyclic nucleotides may act as signal-bearing molecules through cell surface cyclic nucleotide receptors (32) or by directly interacting with ion channels (29). Thus the function of cyclic nucleotide egression would be more than just a means for controlling intracellular cyclic nucleotide concentration. The present study showed that cGMP produced by ATII cells in response to ANP was predominantly secreted across the basolateral membrane. This observation is in accord with a previous report (10) on human airway epithelial cells showing that CNP stimulation resulted in a predominant basolateral extrusion. The polarity of cGMP egression in epithelial cells is likely dependent on the cell type because in two types of renal cells, podocytes and principal cells of the collecting duct, cyclic nucleotides were preferentially released at the apical (luminal) pole (1). This polarized extrusion might reflect a specific biological role for cGMP in the extracellular compartment, such as modulation of phosphodiesterase activity or transport process (9). The nature of the transport system(s) responsible for the efflux of cGMP has not been established. The transporter exhibits some properties of organic anion transporter, especially probenecid sensitivity. In ATII cells, ANP-dependent cGMP egression from the basolateral pole of the cells was inhibited by probenecid, whereas apical egression was not significantly affected, suggesting the prevailing basolateral localization of the probenecid-sensitive transporter, in agreement with the study by Geary et al. (10) on airway human cells. GC iself, which exhibits topologic similarities with membrane channels or transporters (16), could account for the probenecid-insensitive part of the cGMP efflux as proposed for endothelial cells (13).
Besides GC receptors, the other class of ANP receptors corresponds to
the clearance receptors that exert an important function of
degradation. Generally, clearance receptors are by far the most
abundant class of ANP receptors in target cells of natriuretic peptides
(21). Clearance receptors could be a characteristic of ATII cells that
would be lost in culture. Although this hypothesis cannot be excluded,
it seems unlikely because expression of clearance receptors usually
increases dramatically in culture conditions (31). In the present
study, the lack of effect on
125I-ANP binding of C-ANP-(423),
a specific ligand of ANP clearance receptors, strongly suggested that
ATII cells did not express this receptor subtype. However, a previous
study has established that clearance-receptor expression
in tissues and cultured cells varied according to the temperature;
clearance receptors were almost undetectable at 0°C, whereas they
were the predominant ANP binding protein at 37°C (2). In our study,
competitive binding studies were performed at 4°C because ATII
cells rapidly degraded 125I-ANP at
37°C, raising the possibility that the lack of expression of
clearance receptors was the consequence of experimental conditions. This hypothesis was ruled out by the results of RT-PCR, which showed
that clearance-receptor mRNA was not detected in ATII cells, in
contrast to mesangial cells. In the absence of clearance receptors, other processes such as membrane ectoenzymes can be at the origin of
ANP degradation by ATII cells. In vivo studies have concluded that
aminopeptidase (25) and neutral endopeptidase (19) hydrolyzed ANP in
lungs. Because neutral endopeptidase is largely expressed in ATII cells
(19), such a mechanism is likely to play a major role in ANP
degradation by these cells.
The physiological role of ANP on the alveolar epithelium has not yet been elucidated. Our findings indicated that ANP inhibited amiloride-sensitive 22Na influx in cultured ATII cells. This inhibitory effect was concentration dependent, occurring only for 100 nM ANP, a concentration that induced a cGMP accumulation of 10 times that of the basal value. These results are in-line with previous studies in renal cells showing that ANP and cGMP inhibit Na uptake (24). Conceivably, ANF reduced Na influx in ATII cells in a fashion similar to that described for kidney epithelium (18). In the kidneys, ANP binds to its epithelial receptors and inhibits the Na channel by activating a cGMP-dependent protein kinase or by modulating G proteins that directly inhibit the channel or activate G protein-dependent kinase (18). In our experimental conditions, ANP did not directly modify Na-K-ATPase activity, which suggested that the effect of ANP on Na channels was relatively specific. The local pulmonary consequences of this effect of ANP on Na transport from the alveolar space remain to be evaluated. In-line with our data, a recent report (28) showed that in isolated perfused liquid-filled rat lungs, ANP infused through the pulmonary circulation decreased Na transport from the alveolar lumen and increased the alveolar permeability. Because, during cardiac dysfunction, there is an increase in lung ANP production, the role of ANP to limit alveolar edema has been evoked. The latter study and our own data suggest that the preventive role of ANP on pulmonary edema is not explained by its direct effects on alveolar epithelial cells but is rather a consequence of its vasodilatory effects on the pulmonary vasculature.
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ACKNOWLEDGEMENTS |
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We thank Y. Savitch for technical assistance.
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FOOTNOTES |
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This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the Facultés de Médecine Xavier-Bichat et Saint-Antoine.
Address for reprint requests: C. Clerici, Laboratoire de Physiologie, UFR de Médecine, 74 rue Marcel Cachin, 93012 Bobigny Cedex, France.
Received 27 March 1997; accepted in final form 4 November 1997.
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