ELSEVIER
The H295R human adrenocortical cell line contains functional atrial natriuretic peptide receptors that inhibit aldosterone biosynthesis
V. Bodarta,b, W.E. Rainey®, A. Fournierd, H. Ongb, A. De Léana,*
a Department of Pharmacology, Faculty of Medicine, Université de Montréal, C.P. 6128 succursale Centre-Ville, Montréal, Québec H3C 3J7 Canada
Faculty of Pharmacy, Université de Montréal, Montréal, Québec H3C 3J7 Canada
“Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center Dallas, TX 75235, USA INRS-Santé, Pointe-Claire, Québec H9R 1G6, Canada
Received 4 October 1995; accepted 29 January 1996
Abstract
The inhibitory effect of atrial natriuretic peptide (ANP) on angiotensin II (AII)-stimulated aldosterone secretion has been previously studied in rat and bovine adrenal zona glomerulosa cells in primary culture. However the understanding of the mode of action of ANP at the molecular level has been hampered by limitations of those primary cell culture systems and by the lack of cell lines from human adrenal cortex. Here we demonstrate the presence of fully functional ANP receptors in the recently characterized AII-responsive adrenocortical carcinoma cell line H295R. Specific saturable binding of 125I-rANP to H295R cell membrane preparations revealed a single class of high affinity binding sites with a density of 20 fmol/mg of protein. The pharmacological profile of this ANP receptor was documented by competitive binding of 1251-rANP with naturally occurring natriuretic peptides. rANP was the most potent with a Ka of 42 pM. pBNP32 was less potent with a Ka of 174 pM. 125I-rANP binding was not competed by pCNP (NPRB-specific ligand) nor by C-ANF (NPRC-specific ligand). Photoaffinity labeling of membrane preparations with 125I-BPA-ANP revealed a single specific protein of molecular weight around 130 kDa. This protein was further identified by immunodetection with a specific antibody directed to the human ANP-specific receptor NPRA. Natriuretic peptides stimulated cGMP production by the receptor-coupled guanylate cyclase with the same specificity. Aldosterone production by AII-stimulated H295R cells was dose-dependently inhibited by rANP with an ED50 of 1.5 nM. In addition, we used this model to test two chimeric analogs of ANP and BNP. pBNP1 and pBNP3 were, respectively, 4- and 2-fold more potent than rANP in competing for 1251-rANP binding with Ka of 10 and 20 pM. pBNP1 was 24-fold more potent in inhibiting AII-stimulated aldosterone production with ED50 of 63 pM. pBNP1 is therefore the most potent natriuretic peptide analog tested. In summary, the human H295R cell line contains NPRA receptors positively coupled to the particulate guanylate cyclase and that antagonize angiotensin II stimulation of aldosterone secretion.
Keywords: Natriuretic peptide; Receptor; H295R cell (human)
1. Introduction
Angiotensin II (AII) is considered as the main physi- ological hormone regulating blood pressure, fluid and electrolyte homeostasis. In the adrenal cortex, AII acutely stimulates aldosterone production that results in increased sodium retention and increased blood vol-
ume. The cardiac hormone atrial natriuretic peptide (ANP) acts in many target tissues to oppose the actions of AII. Thus ANP inhibits aldosterone production in- duced by AII in the adrenal cortex. Excessive aldos- terone secretion is manifested by hypertension, whereas deficient production leads to salt wasting characteristic of Addison’s disease. Knowledge of the regulatory mechanisms of aldosterone secretion is a prerequisite to understand the etiology of these disorders and to de- velop therapeutic treatments.
* Corresponding author. Tel .: + 1 514 343 6334; Fax: + 1 514 343 2291; E-mail: delean@ere.umontreal.ca.
The inhibitory effect of ANP on AII-stimulated al- dosterone production has been previously studied in rat, bovine and human adrenal zona glomerulosa cells in primary culture [1-3]. However, the understanding of the mode of action of ANP at the molecular level has been hampered by limitations of those primary cell culture systems. Long-term culture of these cells leads to progressive loss of responsiveness to AII and ANP, and to decaying aldosterone production [4,5]. The con- stant requirement for freshly acquired tissue along with the lack of readily available human tissue and the difficulties associated with the isolation of zona glomerulosa cells are major obstacles to defining the molecular mechanism of ANP inhibition of aldosterone secretion.
Recently, a human adrenocortical carcinoma cell line (H295R) has been reported in the literature [6]. This human cell model system secretes low amounts of al- dosterone under basal conditions but interestingly re- tains functional control of aldosterone production by AII [7]. The purpose of this study was to characterize the natriuretic peptide receptors present in the H295R cell line. Three natriuretic peptide receptor classes have been described. Receptors of the NPRA and NPRB classes possess an intrinsic guanylate cyclase catalytic activity. ANP and brain natriuretic peptide (BNP) have similar high affinity for the NPRA receptor subtype while the C-type natriuretic peptide (CNP) has high affinity for the NPRB receptor subtype. The third class of receptor, NPRC, does not discriminate between these peptides and recognizes biologically inactive ANP derivatives, e.g., [Cys116]rANP-(102-116)NH2 (C- ANF). In vitro and in vivo studies indicate that the regulation by natriuretic peptides of aldosterone pro- duction is mediated by NPRA receptors [8,9]. Herein we report that the human H295R cell line possesses fully functional natriuretic peptide receptors. These re- ceptors are of the NPRA subtype, are positively cou- pled to the particulate guanylate cyclase and inhibit AII-stimulated aldosterone production. In addition, we used this human cell model as a bioassay to test syn- thetic analogs of natriuretic peptides. These peptides proved to be significantly more potent inhibitors of aldosterone production than the naturally occurring natriuretic peptides.
2. Materials and methods
2.1. Materials
Dubelcco’s Modified Eagle’s-Ham’s F-12 medium (DMEM/F12) was purchased from Gibco Labs Inc. (Burlington, Ontario). Nu-Serum serum replacement and ITS + Premix Universal Culture supplement were from Collaborative Biomedical Products Inc. (Bedford,
MA). Angiotensin II, rat ANP, porcine BNP32, porcine CNP and [Cys116]ANF(102-116)-NH2 (C-ANF) from Peninsula (Belmont, CA). Natriuretic peptide chimeric analogs pBNP1 and pBNP3 were synthesized according to Mimeault et al. [10]. The peptide [Tyr116, p-benzoyl- L-Phe125JANP(102-125) (BPA-ANP) was obtained from IAF Biochem (Montréal, Québec). Iodo-Beads were from Pierce Chemicals Co. (Rockford, IL), and carrier-free Na125I was from Amersham (Oakville, On- tario). 125I-rANP and 125I-BPA-ANP were prepared by radioiodination of rANP and BPA-ANP using the solid-phase Iodo-Beads method [11]. cGMP and ScGMP-TME were purchased from Sigma Chemical Co. (Saint-Louis, MO). Anti-cGMP antibody was kindly provided by Dr A. Bélanger (Centre Hospitalier de l’université Laval, Québec, Canada). 1251-cGMP was prepared by radioiodination of 2’-O-monosuccinyl- cGMP tyrosylmethyl ester using the solid-phase Iodo- Beads method [12]. All monoiodinated compounds were purified on a reverse-phase Vydac C18 column. Aldosterone-3-(O-carboxymethyl)oximino-(2-[125I]iodo- histamine) was purchased from Diagnostic Products Corporation (Markham, Ontario). Anti-aldosterone-3- BSA-antibody was from ICN Biomedicals Inc. (Costa Mesa, CA).
2.2. Cell culture
H295R cells were selected from the NCI-H295 cell line obtained from the American Type Culture Collec- tion (ATCC, Rockville, MD) as previously reported [7]. The cells were maintained in a 1:1 mixture of Dubelc- co’s Modified Eagle’s and Ham’s F-12 media (DMEM/ F12) containing pyridoxine HCI, L-glutamine and 15 mM Hepes, and supplemented with insulin, transferrin, selenium (1% ITS +), 2.5% Nu-Serum and antibiotics. Cells were grown in 75-cm2 flasks at 37°℃ under an atmosphere of 5% CO2-95% air. Where aldosterone and cGMP production were studied, cell monolayers were subcultured and after 48 h, medium was replaced with fresh serum-free medium (DMEM/F12 containing 0.01% BSA). Cells were cultured for a further 24 h, then rinsed and treated in the same medium. At the end of the incubation, the medium was removed and stored frozen at - 20°℃ for subsequent assay.
2.3. Preparation of membranes
The H295R cells were grown as monolayers as de- scribed above. Confluent H295R cells were washed three times in ice-cold saline and detached with a rubber policeman. The cells were homogenized in buffer containing 20 mM NaHCO3, 2 mM EDTA, 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 uM aprot- inin, 1 µM leupeptin and 1 uM pepstatin A. After
centrifugation, the pellet was washed twice, resus- pended in buffer containing 50 mM Tris-HCI pH 7.4, 250 mM sucrose, 0.1 mM EDTA and frozen in liquid nitrogen. Membranes were stored at - 80℃ until used.
2.4. Receptor binding assay
Membranes (20 µg) were incubated with 6-8 pM 125I-rANP and varying concentrations of unlabeled competing peptides. The binding assay was carried out at 22℃ for 90 min in 1 ml of buffer containing 50 mM Tris-HCI pH 7.4, 0.1 mM EDTA, 0.5% BSA and 5 mM MnCl2 (or 5 mM MgCl2 when C-ANF was used as competitor). For saturation curves, membranes (40 µg) were incubated with increasing concentrations of 125I- rANP (5-300 pM) in 0.2 ml of binding buffer. Mem- brane-bound 1251-rANP was separated from free ligand by filtration through 1% polyethyleneimine-treated GF/ B filters, followed by extensive washing with 50 mM phosphate buffer pH 7.4. 1251-rANP retained on the filters was counted in a / counter.
2.5. Photoaffinity labeling with 1251-BPA-ANP
Photoaffinity labeling of H295R cell membranes was done according to McNicoll et al. [13]. Briefly, mem- branes (200 µg) were incubated with 60 pM 125I-BPA- ANP in the presence or absence of 0.1 AM rANP in 1 ml of binding buffer for 90 min at 22℃. At the end of the incubation time, the tubes were placed on ice at a distance of 8 cm from two mercury lamps (54 mW cm2 at 365 nm). After 20 min irradiation, the tubes were centrifuged and the pellets were washed twice with buffer containing 50 mM Tris-HCI, pH 7.4, and 0.1 mM EDTA. All steps were done in the darkness. The photolabeled membranes were boiled for 5 min in sample buffer and subjected to 5% SDS-PAGE elec- trophoresis. The gel-fractionated proteins were trans- ferred to nitrocellulose membrane by the semidry blotting method using a Multiphor II Novablot Elec- trophoretic Transfer Unit (LKB) according to manu- facturer’s instructions. Autoradiographic exposure was done at - 80℃ with a Dupont NEN Reflection inten- sifying screen and Dupont Reflection autoradiographic film.
2.6. Immunodetection
The antibody used for immunodetection was devel- oped against the synthetic peptide CS368 (Y-G-E-R-G- S-S-T-R-G) whose sequence was based on the carboxy-terminal sequence Gly1021-Gly1029 of the hu- man NPRA receptor flagged with a tyrosine at its amino-terminal end. The peptide was coupled to BSA using glutaraldehyde and 100 µg of conjugate was injected subcutaneously to rabbits. Antisera were col-
lected after three boots. The immunoglobulin was purified by affinity on a CS368-agarose column before immunoblotting was performed.
After autoradiography of the photolabeled proteins, the nitrocellulose membrane was blocked in PBS con- taining 0.1% Tween and 5% dry milk overnight at 4℃. The membrane was washed three times in PBS contain- ing 0.1% Tween and incubated with anti-NPRA anti- body diluted in PBS containing 0.1% Tween and 0.1% BSA for 90 min at 22°C. After the incubation time, the membrane was washed three times in PBS containing 0.1% Tween and incubated with anti-rabbit Ig horseradish peroxidase-linked antibody for 60 min at 22℃. Then the membrane was washed 5 times in PBS containing 0.1% Tween and the proteins were detected by the enhanced chemiluminescence method according to manufacturer’s instructions (ECL kit, Amersham).
2.7. Aldosterone determination
Aldosterone was directly measured in cell culture medium by a specific radioimmunoassay as already described [14]. The least detectable concentration mea- sured by the RIA was 5 fmol/ml.
2.8. Cyclic GMP determination
H295R cells were incubated in DMEM/F12 medium in the presence of the phosphodiesterase inhibitor 0.5 mM 3-isobutyl-1- methylxanthine (IBMX) and extracel- lular cyclic GMP production was measured by radioim- munoassay as already described [15].
2.9. Data analysis
Radioligand binding saturation curves and radioli- gand binding competition curves were analyzed by the program SCAFIT based on the law of mass action [16]. The affinity of peptides for NPRA is expressed as pK; or log K where K is the affinity constant in M units. Dissociation constants Ka = 1/K are also reported. Dose-response curves for cGMP production and aldos- terone inhibition were analyzed with the ALLFIT for Windows1 program based on a four-parameter logistic equation in order to obtain estimates of the ED50 [17]. Statistical differences were tested by ANOVA and Bon- ferroni multiple comparison t-test after checking for homogeneity of variance. Statistical significance levels were set at P<0.05 (significant) and P <0.01 (highly significant).
’ Requests for the program Allfit for Windows can be addressed by E-mail to: delean@ere.umontreal.ca.
3. Results
3.1. Pharmacological characterization of natriuretic peptides receptors in H295R cell membranes
The presence of ANP receptor in the human adreno- cortical cell line H295R was demonstrated by specific saturable binding of 125I-rANP to membrane prepara- tions. Computer analysis of the binding of radiolabeled ANP over the concentration range tested revealed a single class of saturable high affinity binding sites with a pK; of 9.96 (Ka = 110 pM) and a Bmax (density) of 20 fmol/mg of protein (Fig. 1). The pharmacological profile of this ANP receptor was established by compet- itive binding of 125I-rANP with different peptides (Fig. 2). Co-incubation of radiolabeled ANP with increasing concentrations of unlabeled rANP resulted in a concen- tration-dependent inhibition of binding with a pK; of 10.39 ±0.15 (Ka=41 pM). The porcine brain natri- uretic peptide (pBNP32) also competed for 125I-rANP binding; however a 4-fold higher concentration was required to produce this effect (pK; = 9.77 ± 0.07, Ka = 170 pM). The porcine C-type natriuretic peptide (pCNP), which is specific for the subtype NPRB, did not inhibit the binding of 125I-rANP to H295R cell membranes even at the highest dose tested. The peptide C-ANF is a highly selective ligand for the NPRC subtype (clearance receptor) and did not significantly
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compete with 125I-rANP binding in H295R cell mem- branes suggesting the absence of the NPRC subtype in H295R cells (Fig. 2). Thus the ANP receptors present in H295R cells showed the typical pharmacological profile of the NPRA subtype: ANP > BNP >> CNP.
Then we used this human adrenocortical cell model to test two chimeric analogs of natriuretic peptides. These peptides were obtained by substituting the amino-terminal (pBNP3) or both carboxy- and amino- terminal (pBNP1) segments of pBNP32 with the corre- sponding exocyclic segments of rANP [10]. As shown in Fig. 3, both peptide analogs exhibited higher affinity for NPRA receptor than did the parent compounds. pBNP1 and pBNP3 were, respectively, 4- and 2-fold more potent than rANP in competing for 125I-rANP binding with pK; values of 11.00 ± 0.12 (Ka= 10 pM) and 10.71 ± 0.04 (Ka=19 pM).
3.2. Biochemical characterization of natriuretic peptides receptors in H295R cell membranes
Photoaffinity labeling of H295R cell membranes with 125I-BPA-ANP resulted in the detection of a major radioactive band of approximately 130 kDa (Fig. 4A). The labeling of this band was inhibited by an excess of rANP (0.1 µM). The corresponding protein was iden- tified as the NPRA receptor by immunodetection with a specific antibody directed to the carboxy-terminal tail of the human NPRA receptor (Fig. 4B).
3.3. Effects of natriuretic peptides on cGMP production in H295R cells
Cyclic GMP produced by the ANP receptor-coupled guanylate cyclase was measured in the incubation medium in the presence of the phosphodiesterase in- hibitor IBMX. rANP and pBNP32 stimulated cGMP synthesis in H295R cells in a concentration-dependent manner with an ED50 of 12.64 ± 2.96 and 15.76 ± 2.39 nM respectively (Fig. 5). As suggested by its higher affinity for the NPRA receptor, pBNP1 was more po- tent than rANP in stimulating cGMP synthesis with an ED50 of 1.67 ±0.91 nM. Even at the highest concentra- tion tested, pCNP had no effect on cGMP production in H295R cells confirming the absence of the NPRB subtype in these cells (Fig. 5).
3.4. Effects of natriuretic peptides on aldosterone production in H295R cells
The human adrenocortical cell line H295R synthe- sized aldosterone in response to AII. As shown in Fig. 6, the peptide rANP inhibited dose-dependently AII- stimulated aldosterone production with an ED50 of 1.5 nM. pBNP32 and pBNP1 also inhibited AII-stimulated aldosterone production with the same order of potency as for receptor binding and for cGMP production. The ED50 values of pBNP32 and pBNP1 were 3.4 and 0.06 nM respectively (data not shown).
I-rANP bound (pM)
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In addition to AII, we have tested three other secret- agogues of aldosterone in the human adrenocortical H295R cell line. High extracellular potassium concen- tration (16 mM) stimulated aldosterone production more profoundly than AII in these cells (4.1-fold vs. 3-fold). But K + -stimulated aldosterone production was inhibited to a lesser extent than the AII-stimulated one by rANP (33.4 vs. 70.7%). Forskolin which activates intracellular cAMP production, could also stimulate 4-fold aldosterone synthesis in H295R cells. In this case, rANP inhibition was not significant. Thapsigargin which increases the cytosolic free calcium concentration by inhibiting the microsomal calcium ATPase-pump, stimulated aldosterone production by H295R cells 3.6- fold. In this case, rANP inhibited aldosterone produc- tion by 53.7% (Fig. 7).
4. Discussion
We report in this paper the presence in the human adrenocortical H295R cell line of fully functional ANP receptors that inhibit secretagogue-stimulated aldos- terone synthesis. Competition binding studies revealed
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a single class of binding sites with a pharmacological profile characteristic of the NPRA receptor subtype: ANP ≥ BNP >> CNP. This is the first report of binding
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CTL AII KCI FORSK THAPS
affinities of natriuretic peptide receptor in isolated hu- man adrenocortical cells. The pharmacological profile of the NPRA receptor present in H295R cells strongly correlates with that of the cloned human NPRA recep- tor. The pK; values obtained by Bennett et al. [18] with NPRA extracellular domain-IgG fusion protein were 11.7 for hANP, 11.1 for hBNP32 and <6.3 for CNP.
The human H295R ANP receptor was further char- acterized by photoaffinity labeling. As in bovine zona glomerulosa cells, the ANP receptor appeared as a 130 kDa labeled protein. The specificity of the photolabeled receptor protein was demonstrated by direct competi- tion of 1251-BPA-ANP with rANP. In addition, no specific photolabeled protein was present in the range of 60-70 kDa confirming the absence of significant amount of NPRC in human adrenocortical H295R cells.
The functionality of the NPRA receptor present in H295R cells has been assessed by measuring cyclic GMP production in response to various natriuretic peptides. rANP, pBNP32 and pCNP stimulated cGMP production with the same rank order of potency as that for receptor binding (ED50 - 12.64 and 15.76 nM for rANP and pBNP32, respectively). pCNP was inactive even at the highest concentration tested confirming the absence of the CNP-specific NPRB receptor subtype in H295R cells. Once more, the ED50 values correlate well with those reported for the cloned human NPRA recep- tor [19]. More importantly, natriuretic peptides inhib- ited aldosterone synthesis evoked by angiotensin II in
human adrenocortical H295R cells. Once more, rANP was more potent than pBNP32 and pCNP had no effect. The ED50 value obtained (1.5 nM for rANP) was commensurate with that observed in bovine and rat zona glomerulosa cells (ED50 = 0.12 and 0.17 nM, re- spectively) [1,2]. Furthermore rANP inhibited to vari- ous extent aldosterone production stimulated by well-known secretagogues like KCI and thapsigargin. In contrast, rANP had no significant effect on forskolin- stimulated aldosterone production. Additional studies on this differential effect of ANP on agonist-stimulated aldosterone production could shed light on its mode of action.
Natriuretic peptide receptors have been characterized in other adrenal cell lines. Mizuno et al. [20] reported the presence of specific ANP receptors in the human adrenal tumor cell line SW-13. These receptors were in very low amount because they could only be detected by their stimulatory effect on cGMP production and not by binding with 125I-rANP. Moreover, the pharma- cological profile was different from that observed in the H295R cell line. rANP and pBNP both increased cGMP production in SW-13 cells but pBNP was con- siderably less potent than rANP suggesting that, in these cells, the ANP receptor could be a distinct NPRA receptor isoform. The biological activity of these ANP receptors on steroid production was not investigated. In addition, the SW-13 cell line produces undetectable amount of aldosterone under basal conditions and this production cannot be stimulated by AII limiting the use of this cell line for investigating the hormonal control of aldosterone secretion [21].The Y-1 mouse adrenocor- tical tumor cell line has been tentatively used as a model to define the site of action of ANP in the steroid biosynthetic pathway, despite its lack of aldosterone and cortisol biosynthesis [22]. These cells possess spe- cific binding sites for ANP. But, in contrast with the H295R cells which express only NPRA receptors, both NPRA and NPRC receptors are present in Y-1 cells. In addition, the binding affinities of these receptors are 25-fold weaker than that observed in H295R cells. Another weakness of this model is the absence of AII stimulation of steroid production.
The human adrenocortical H295R cell line could also be useful for the screening of synthetic natriuretic pep- tide analogs. Compounds that are more selective for the NPRA receptor than the naturally occurring ones could be of therapeutic interest in the treatment of hyperten- sive disorders. Mimeault et al. have characterized in bovine adrenal cortex and rat papillae two synthetic chimeric analogs of natriuretic peptides (pBNP1 and pBNP3) that had higher affinities for the NPRA recep- tor subtype than rANP, but possessed affinities com- parable to that of rANP for the NPRC receptor [10]. Therefore, these peptides could be more effective than the natural hormone ANP. However, the biological
effect of these analogs on aldosterone production had never been tested. In our human adrenocortical H295R cell line model, pBNP1 showed a 4-fold higher affinity for the NPRA receptor than rANP and was 7.5-fold more potent than rANP to stimulate cGMP produc- tion. More interestingly, the peptide pBNP1 was 24- fold more potent than rANP to inhibit AII-stimulated aldosterone production. Therefore, pBNP1 is the most potent natriuretic peptide analog tested. Its higher po- tency over naturally occurring peptides is especially prominent on the physiologically meaningful function of aldosterone production. Current works in progress will document the structural properties of this highly potent pBNP1 analog and will attempt to further im- prove its potency and its specificity for NPRA recep- tors.
In conclusion, the human adrenocortical H295R cell line, as far as we have investigated, is a model at all points similar to the bovine zona glomerulosa cells in primary culture. The H295R cell line provides an in- valuable model for defining the molecular mechanism of action of ANP leading to adrenal steroidogenesis inhibition and testing synthetic analogs of potential therapeutic interest.
Acknowledgements
This work was supported by a program grant from the Medical Research Council of Canada and a grant from the Kidney Foundation of Canada. A.D.L. is a recipient of a PMAC-MRC research chair in Pharma- cology sponsored by Merck-Frost Canada. We thank Normand McNicoll for his stimulating discussions and judicious advice.
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