Characterization and regulation of atrial natriuretic peptide (ANP)-R1 receptors in the human neuroblastoma cell line NB-OK-1

European Journal of Pharmacology - Molecular Pharmacology Section, 207 (1991) 81-88

81

© 1991 Elsevier Science Publishers B.V. 0922-4106/91/$03.50 ADONIS 092241069100111W EJPMOL 90179

Characterization and regulation of atrial natriuretic peptide (ANP)-R~ receptors in the human neuroblastoma cell line NB-OK-1 Christine Delporte, Piotr Poloczek, Denis Gossen, Michrle Tastenoy, Jacques Winand and Jean Christophe Department of Biochemistry and Nutrttton, Medtcal School, Unioersit~ Libre de Bruxelles, B-IO00 Brussels. Belgium

Received 31 October 1990, revised MS recewed 9 January 1991, accepted 12 February 1991

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R t receptor (Mr 130 kDa) for the atrial natriuretic peptide (ANP). This receptor recognized biologically active forms of ANP with high affinity but showed no affinity for truncated ANP forms. It was functional in that binding correlated with guanylate cyclase activation (a 2-fold increase in Vmax) with the following rank order of potency: rat ANP-(99-126) > human ANP-(99-126) > human ANP-(102-126) > porcine BNP (brain natriuretic peptide). The enzyme required free Mn 2÷ in addition to the Mn-GTP substrate (K m of about 0.3 mM for both basal and ANP-stimulated activity). In the presence of dithiothreitol, the dose-response curve of guanylate cyclase activation was shifted rightward by a factor of 30. ANP-R 1 receptors were upregulated through protein synthesis in cells exposed to 1 mM carbamylcholine or 1 mM dibutyryl cyclic AMP for 8-24 h (ANP was ineffective). ANP-R~ receptors; Guanylate cyclase; Neuroblastoma cell line NB-OK-1; (Human

1. Introduction

The peptidic hormone atrial natriuretic peptide ( A N P ) produces several biological effects including diuresis, natriuresis, vasorelaxation and inhibition of aldosterone secretion (for review see Genest and Cantin, 1988; Inagami, 1989). In rat, A N P is produced by atria as a 126 amino acid precursor (pro-ANP) and is processed to the major circulating form ANP-(99-126) (Thibault et al., 1985). Three types of A N P receptors are recognized at present. The A N P - R 1 type of receptor has a M r of 130 k D a which is not decreased by reduction. It exhibits selectivity for ANP-(99-126) over ANP-(103-123) and possesses intrinsic guanylate cyclase activity (Leitman et al., 1988; Scarborough et al., 1986; Takayanagi et al., 1987). The A N P - R 2 receptor has a M r of 130 k D a under non-reducing conditions that is decreased to 64-70 k D a in the presence of reducing agents. It lacks guanylate cyclase activity, shows no selectivity for ANP-(99126) and may assume a clearing function (Maack et al., 1987). A third A N P receptor (R3) was recently delineated by two groups after screening c D N A Libraries of h u m a n placenta (Chang et al., 1989) and rat brain

Correspondence to: J. Chrtstophe, Department of Biochemistry and Nutrition, Medical School, Umversit6 Libre de Bruxelles, Boulevard of Waterloo 115, B-1000 Brussels, Belgmm.

(Schulz et al., 1989). This receptor ts structurally similar to A N P - R t with more than 70% homology in the region coding for the intracellular tail (kanase-like and guanylate cyclase domains). This A N P - R 3 receptor, with a predicted M r of 115 kDa, is preferentially occupied by porcine brain natriuretic p e p t i d e (BNP) rather than by ANP-(99-126) and, like the A N P - P t receptor, is endowed with intrinsic guanylate cyclase activity (Chang et al., 1989; Schulz et al., 1989). Brain areas involved in cardiovascular and fluid homeostasis contain A N P - l i k e immunoreactivity as well as A N P receptors (Kurihara et al., 1987; Saavedra et al., 1986; Z a m i r et al., 1986), which may account for some neuroendocrine actions of A N P . In addition, the parent peptide BNP exists as a 32 amino acid peptide in rat and h u m a n brain and as a N-terminally shortened isoform of 26 amino acids in pig brain (Sudoh et al., 1988, 1990). Because of the interest in elucidating the role of A N P (and BNP) as neurotransmitter or n e u r o m o d u l a t o r in the central nervous system, we tested the presence of functional A N P / B N P receptors in the human neuroblastoma cell line NB-OK-1. This cell line has receptors for VIP and P A C A P (Cauvin et al., 1990) and muscarinic receptors (Waelbroeck et al., 1988). It also synthesizes VIP and P H M from p r o - P H M / V I P (Itoh et al., 1983; Svoboda et ai., 1986). The aim of our study was to characterize: (1) A N P receptors in binding and crosslinking experiments; (2) the associated m e m b r a n o u s

82 guanylate cyclase activity; (3) the regulation of ANP receptor expression. Part of these data have been published in abstract form (Delporte et al., 1990).

ml/min with a 10-50% linear gradient of solvent B (80% CH3CN-0.1% TFA) in solvent A (5% CH3CN-0.12% TFA). With this system, the retention time of [125I]ANP-(99-126) was 11.5 rain.

2. Materials and methods

2.5. Guanylate cyclase assay

2.1. Cell culture and membrane preparation

Measurement of enzyme activity was performed by preincubating membrane aliquots for 10 rain in a total volume of 60/~l containing 1 mM GTP, 3 mM MnC12, 1 mM guanosine 3':5'-cyclic monophosphate (cyclic GMP), 1 mM 3-isobutyl 1-methylxanthine (IBMX), 10 mM creatine phosphate, 1.2 U / a s s a y phosphocreatine kinase, 0.5% (w/v) bovine serum albumin BSA and 50 mM Tris-HCI at a final pH of 7.6. The reaction was initiated by addition of 1 /~Ci of [a-32P]GTP in 10/~1 and was terminated after 15 min of incubation at 3 7 ° C by adding 300 /~1 of 1 M HCIO4 containing 50000 cpm [8-3H]cyclic GMP. Cyclic GMP was separated from GTP by two successive chromatographies on Dowex 50-WX8 and neutral alumina (White and Karr, 1978).

NB-OK-1 cells were grown in RPMI 1640 medium enriched with 10% fetal calf serum and antibiotics. At confluence the cells were detached with a rubber policeman, centrifuged for 10 min at 100 × g, rinsed with the culture medium, lyzed in 1 mM NaHCO 3 then quickly frozen in liquid N 2. The lysate was defrosted and centrifuged at 4 ° C at 100 × g for 10 min. The supernatant was centrifuged at 20000 × g for 10 min. The pellet was rehomogenized in 1 mM NaHCO 3 and aliquots of this crude membrane preparation were immediately tested.

2.2. Peptide radioiodination Rat ANP-(99-126) was radioiodinated on the Cterminal tyrosine by the iodogen method and purified on a C18/tBondapak column (Fraker and Speck, 1978). Tracer specific activity was 1900-2000 Ci/mmol.

2.3. Binding assay Membranes were incubated at 37 ° C in a total volume of 120 /~1 containing 25 mM Tris-HCl, 2 mM MgCI2, 0.4% (w/v) bovine serum albumin, 500 KIU (kallikrein inhibitor units)/ml Trasylol, 0.1 mg/ml bacitracin (pH 7.4) and rat [125I]ANP-(99-126) in the 5000 to 200000 cpm range (for saturation experiments) or with 20000 cpm/assay in standard binding conditions, that corresponded to a final tracer concentration of 40 pM. Non-specific binding was determined in the presence of 0.3 /xM ANP-(99-126). The separation of membranebound and free radioactivities was achieved by rapid filtration through glass-fiber filters (Whatman G F / C ) presoaked for 24 h in 0.1% poly(ethyleneimine).

2.6. Cross-linking of [I"51]ANP-(99-126) Membranes were incubated for 20 rain at 37 ° C with 40 pM [125I]ANP-(99-126) in the medium used for the binding assay. Membranes were washed twice by centrifugation and resuspension in ice-cold PBS (50 mM phosphate buffer (pH 7.4) containing 150 mM NaC1) and resuspended in the same buffer. Disuccinimidyl suberate (DSS, 0.2 mM or 0.4 mM final concentration) was added to the membrane suspension. After a 45 min incubation at 4 ° C, the membranes were washed twice in ice-cold PBS then solubilized in an electrophoresis sample buffer made of 125 mM Tris-HC1 (pH 6.8) containing 5% (w/v) SDS, 10% (w/v) sucrose, 0.02% (w/v) bromophenol blue and, where indicated, 1% (w/v) dithiothreitol (DTT) and 4% (w/v) 2-mercaptoethanol. After heating for 2 min at 100°C, samples were submitted to SDS-PAGE under reducing or non-reducing conditions with a 12% homogeneous polyacrylamide separating gel (180 × 200 x 1.5 mm). Autoradiographies were conducted for 3 wk at - 8 0 ° C.

2.4. Degradation of [t'-51]ANP-(99-126) 2.7. Protein determination Membranes were incubated as described in section 2.3 for 20 min with 40 pM tracer. After rapid centrifugation at 9000 × g, the supernatant was acidified with 0.1% trifluoroacetic acid (TFA) and loaded on a Ca8 Sep-Pak cartridge (Waters, Milford, MA, U.S.A.). After washing with 5% CH3CN-0.12% TFA, radioactive compounds were eluted with 5 ml 80% CH3CN-0.1% TFA. CH3CN was evaporated under nitrogen and the sample loaded on a Nova-Pak C~8 tt-column connected to a Waters-Millipore HPLC gradient system and eluted at 1

Protein determination was measured according to Lowry et al. (1951) using bovine serum albumin as standard.

2.8. Chemicals Rat atrial natriuretic peptide (ANP-(99-126) and rat Atriopeptin I (ANP-(103-123)) were obtained from Novabiochem (Laiafelfingen, Switzerland). Rat ANP-

83 (99-109), rat ANP-(111-126), rat C-ANP-(102-121) (Des[GlnH6,Ser117,GIyH 8,LeuH 9,Gly12° ]-ANP-(102-121)), h u m a n A N P and porcine B N P were purchased from Peninsula (Belmont, CA, U.S.A.). (Des-Cys TM, Cys TM) ANP-(104-126) was a gift from Dr. G.M. Olins (Searle, Stokie, IL, U.S.A.). Carrier-free Na125I (600-800 m C i / m l ) and [83H]cGMP (21 C i / m m o l ) were purchased from Amersham International (Bucks, U.K.) and [a-32p]GTP (800 C i / m m o l ) from New England Nuclear (Boston, MA, U.S.A.). Creatine phosphate, phosphocreatine kinase, N 6 - 2 ' - O - d i b u t y r y l a d e n o s i n e 3 ' : 5'-cyclic m o n o p h o s phate (DBcAMP), guanosine 3 ' : 5 ' - c y c l i c monophosphate, and butyrate were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Bovine serum albumin (fraction V) and G T P were from Boehringer (Mannheim, Germany). Kallikrein inhibitor (Trasylol) was obtained from Bayer (Brussels, Belgium). Fetal calf serum and medium for cell cultures were from Life Technologies Europe (Ghent, Belgium). All other reagents were of the highest analytical grade available.

with an association half-time of 4 min (fig. 1A). N o n specific binding, determined in the presence of 0.3 ~tM A N P , represented 15-20% of specific binding. After a 20 rain preincubati0n with tracer, tracer dissociation implemented by 0.3 # M A N P was biphasic: 22 _+ 1% of the radioactivity dissociated rapidly with a k o , of 1.160 + 0.175 min -1 while 78% dissociated slowly with a k o , of 0.013 + 0.001 min -1 (n = 6) (fig. 1B). The dissociation process was not accelerated by 100 /tM G T P or b y 100 ~ M G p p [ N H ] p . Scatchard transformation of saturation curves with increasing [125I]ANP concentrations was compatible with a single high-affinity class of binding sites exhibiting a K D of 12 _+ 1 p M and a density of 62 +_ 3 f m o l / m g m e m b r a n e protein (n = 9, fig. 1C). In the absence of 0.1 m g / m l bacitracin, the degradation of the tracer increased to 20 _+ 4% (n = 4) after 20 min as c o m p a r e d to only 6+_ 1% in its presence. Scatchard plots of saturation curves with increasing [125I]ANP concentrations after 20 min gave identical results with or without bacitracin. Competition curves (fig. 2) performed for 20 min at 37 ° C in the simultaneous presence of 40 p M tracer and increasing concentrations of unlabelled peptides, showed (after Cheng and Prusoff correction, 1973), the following K, values for rat ANP-(99-126), human ANP-(99126), human ANP-(102-126) and porcine BNP: 17 pM, 30 pM, 55 p M and 186 pM, respectively. The five truncated analogues ANP-(103-123), C-ANP-(102-121), ANP-(111-126), ANP-(99-109) and (des Cys]°5,Cys t21)

Results

3.1. Characteristics of [125I]ANP receptors on neuroblastoma membranes Specific binding of [125I]ANP offered at a 40 p M concentration reached a plateau after 20 rain at 3 7 0 C

cpm 2000-

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Fig. 1. (A) Kinetics of specific [1251]ANPbinding to human neuroblastoma NB-OK-1 cell membranes. Non-specific binding was determined in the presence of 0.3/~M ANP. Total counts in the assays were 20000 cpm. The experiments were performed in duphcate and the results are expressed in cpm_+S.E.M. (n = 4). (B) Time course of [12SI]ANPdissociation from human neuroblastoma NB-OK-1 membranes. Dissociation was studied at 37 °C after 20 min preincubation with 40 pM [12SI]ANP.Dissociation was initiated by the addition of 0.3 p.M ANP. The results are expressed as In specific B/Bmax. Each value was determined in duplicate. This experiment was typical of hve others. (C) Scatchard representation of a saturation curve obtained after incubating increasing [12SI]ANPconcentrations with human neuroblastoma NB-OK-1 cell membranes for 20 min at 37°C. Non-specific binding was determmed in the presence of 0.3/tM ANP. The results are representative of eight others performed in duphcate.

84 A N P - ( 1 0 4 - 1 2 6 ) (the latter three w i t h o u t bridge) did not recognize the receptors.

a disulfide

3.2. Crosslinking of A N P receptors m neuroblastoma membranes A f t e r c o v a l e n t labelling with 0.2 m M D S S (fig 3, lanes 1 and 2) or 0.4 m M D S S (lanes 3 and 4), S D S P A G E , and a u t o r a d i o g r a p h y , a m a j o r specifically labelled b a n d was present u n d e r r e d u c i n g c o n d i t i o n s at 130 k D a with faint additional b a n d s at 90 and 33 k D a (lanes 1 and 3). T h e s e b a n d s were absent (lanes 2 and 4) w h e n the tracer (40 p M [125I]ANP) was c o i n c u b a t e d with 3.10 -7 M A N P . Similar results were o b t a i n e d u n d e r n o n - r e d u c i n g c o n d i t i o n s (not shown).

LANE DSS 0.3 ~M Ab Mr (kOa) 200

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3.3. Parttculate guanylate cvclase activation through A N P receptors M e m b r a n e s were p r e i n c u b a t e d at 37 ° C for 10 min in the m e d i u m w i t h o u t tracer (because of a 2-4 m i n latency phase o b s e r v e d without preincubation), then i n c u b a t e d with [a32P]GTP. T h e r e was a linear relationship between m e m b r a n e protein c o n c e n t r a t i o n and basal or A N P - s t i m u l a t e d g u a n y l a t e cyclase activity in the 1-10 /tg protein range (fig. 4A). Protein c o n c e n t r a t i o n was adjusted accordingly in the following experiments. U n der these conditions, the time course of g u a n y l a t e cyclase activity showed a linear increase in c G M P for at least 30 m m , in the absence as well as in the p r e s e n c e of 1.10 8 M A N P (fig. 4B). A time p e r i o d of 15 min was a d o p t e d in the following experiments. W h e n 0.1 to 5 m M free M n C I 2 c o n c e n t r a t i o n s were tested (fig. 4C), cation c o n c e n t r a t i o n s a b o v e 1 m M allowed the best e n z y m e activity u n d e r b o t h basal and

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-~2-it-io-9 -a -'7 [PEPTIOE) (log M) Fig. 2. CompeUhon curves of ANP analogues for the binding of 40 pM [1251]ANP to human neuroblastoma NB-OK-1 cell membranes for 20 mm at 37 ° C. Non-specific binding was determined m the presence of 0.3 pM ANP. The experiments were performed m duplicate and the results are expressed as percentage B/Bo _+S.E.M. (n = 4). When not shown the error is less than 1%.

30

Fig. 3. Chemical cross-linking of ANP receptors to [1251]ANP with dlsuccinirmdyl suberate. Human neuroblastoma NB-OK-1 cell membranes were incubated with 40 pM [~251]ANP in the presence or absence of 0.3 #M unlabelled ANP for 20 min at 37 o C. Samples were washed twice by centrifugation and resuspension m ice-cold PBS and resuspended m the same buffer before addition of DSS (final concentratlon 0.2 raM, lanes 1 and 2, 0.4 mM, lanes 3 and 4). Samples were submitted to SDS-PAGE under reducing conditions (see Materials and methods). The approximate Mr (× 10 -3) is mentioned on the left Th~s experiment ~s representative of two others.

A N P - s t i m u l a t e d c o n d i t i o n s . In c o n t r a s t C a C I 2 a n d MgC12 were u n a b l e to s t i m u l a t e the e n z y m e in the p r e s e n c e of A N P ( d a t a n o t shown). T h e r e f o r e , in all further e x p e r i m e n t s 2 m M free MnC12 was used. W i t h 0.1 to 5 m M of the M n - G T P substrate, in a d d i t i o n to 2 m M MnC12+, a n d in the a b s e n c e or p r e s e n c e of 1.10 -8 M A N P , m a x i m a l a c t i v a t i o n occurred b e t w e e n 0.1 a n d 1 m M M n - G T P , the L i n e weaver-Burke double reciprocal transformation showing a t w o - f o l d increase in Vmax with A N P ( f r o m 56 _+ 2 p m o l cGMP/min/mg protein to 111 _+ 7 p m o l cGMP/min/mg p r o t e i n ) with no c h a n g e in K m (0.31 _+ 0.04 m M for basal c o n d i t i o n s and 0.25 +_0.02 m M u n d e r A N P s t i m u l a t i o n ) (n = 4, fig. 4D). W i t h 1 m M M n - G T P and 2 m M free MnC12 a n d in the a d d e d p r e s e n c e of 1 m M D ' I T , the d o s e - r e s p o n s e curve of the e n z y m e to A N P was shifted r i g h t w a r d b y a factor of 30 (the ECs0 i n c r e a s i n g f r o m 1.10 -1° M to 3.10 - 9 M, fig. 5).

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Fig. 4. Guanylate cyclase activity. (A) Effects of protein concentration on cGMP generation. Membranes were incubated for 15 rain with 1 mM Mn-GTP and 2 mM free MnCI 2 and with or without 1.10 - s M ANP. Results are representative of three others performed in duplicate. (B) Effects of 1.10 - s M A N P on the time course of guanylate cyclase activity. Membranes were incubated with 1 mM Mn-GTP and 2 mM free MnCI 2 m the presence or absence of 1.10-8 M ANP for time periods up to 30 nun. Experiments were performed in duplicate and results are the means + S.E.M. (n = 4). (C) Effects of increasing free MnCI 2 concentrations on guanylate cyclase activity. Membranes were incubated for 15 rain in the presence of 1 m M Mn-GTP and free MnCI 2 concentrations up to 5 mM. Experiments were performed in duplicate and results are the means + S.E.M. (n = 4). (D) Lineweaver-Burke plot of increasing Mn-GTP concentrations on guanylate cyclase activity, in the added presence of 2 m M MnCI2. Results are representative of three others performed in duplicate.

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ANP-(99-126). BNP was both less potent (ECs0 of 1.10 -9 M as compared to 1.10 -]0 M for ANP) and less effic i e n t (Vma ~ o f 1 4 0 _ 9 p m o l c G M P / m i n / m g protein as c o m p a r e d t o 1 7 0 + 10 p m o l c G M P / m i n / m g protein with ANP). Rat ANP-(103-123), rat ANP-(lll-126), rat

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[RAT ANP-(99-125)] (log M) Fig. 5. Effects of l mM DTT on the response of particulate guanylate cyclase to ANP. Membrane fractions were incubated in the presence or absence of 1 mM DTT and in the presence of 1 mM Mn-GTP, 2 m M free MnCI~ and increasing ANP concentrations. Results are means 5: S.E.M. of four experiments performed in duplicate.

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[RAT ANP-(99-126)] (log M) Fig. 7. ANP-st,mulated guanylate cyclase actiwty in human neuroblastoma NB-OK-1 cell membranes pretreated for 24 h with 1 mM carbamylchohne or 1 mM DBAMPc (Bu2AMPc). The results are expressed m pmol c G M P / m i n / m g protein over basal values and are the means + S.E.M. of four experiments performed in duplicate. Basal activities under control conditions and after carbamylchohne and DBAMPc inductions were, respectively, 70 + 6, 95_+7, and 150 + 10 pmol c G M P / m i n / m g protein.

ANP-(99-109) and rat (des Cysl°5,Cys121) ANP-(104126) were unable to activate the enzyme.

3.4. Regulation of A NP receptor expression , After 8 h ANP receptors increased by 2 8 _ 8% and 16 + 6% (n = 3) in response to carbamylcholine and DBcAMP, respectively. ANP receptors in neuroblastoma cells treated for 24 h with 1 mM carbamylcholine or 1 mM DBcAMP increased by 36 + 11% and 125 + 7% (n --- 5) as compared to control cells and without K D modification. In contrast, 2 mM butyrate and 1.10 -8 M ANP did not influence the density of ANP receptors. Increases in ANP receptors were due to protein synthesis as the presence of 1 /tg/ml cycloheximide blocked the accumulation of extra ANP receptors induced by carbamylcholine or DBcAMP. Increases in receptor number provoked by carbamylcholine and DBcAMP correlated well with increases in both basal and ANP-stimulated guanylate cyclase activities (see fig. 7 and its legend). Figure 7 illustrates the dose-effect curves of rat ANP stimulation after 24 h treatments with 1 mM carbamylcholine and 1 mM DBcAMP.

4. Discussion In the present study we documented the presence of high-affinity ANP receptors in human neuroblastoma NB-OK-1 membranes that seemed to be mostly, if not exclusively, of the ANP-R t type. Indeed truncated ANP

analogues showed no affinity (Fethiere et al., 1989; Olins et al., 1988) and the Mr of the crosslinked site was around 130 kDa in non-reducing as well as in reducing conditions. Scatchard analysis of the saturation curve showed only one class of binding sites that were likely to be functional. The displacement curve of [125I]ANP by BNP was not better fitted with a two-site model than with a one-site model (F2.99; d.f. = 2 and 4; P > 0.10) when analyzed with the non-linear, least-squares curve fitting L I G A N D program (Richardson and Humrich, 1984), suggesting again the absence of ANP-R 3 receptors (see also below). Furthermore, K D values in competitive displacement curves and ECs0 values for guanylate cyclase activation were reasonably well correlated, the rank order of potency of ANP analogues being the same for both parameters: rat ANP-(99-126) > human ANP(99-126) >i human ANP-(102-126) > BNP. All agonists were equally efficacious in activating the enzyme except for BNP (30% less at maximal concentration). Our binding studies were performed in the presence of 0.1 m g / m l bacitracin that largely protected the tracer from degradation without altering its binding to membranes. Tracer dissociation from membranes was biphasic (fig. 1B), a phenomenon possibly due to a small heterogeneity in the ANP-R t receptor population (e.g. by the glycosylation a n d / o r phosphorylation state) a n d / o r in the tracer quality• Cross-linking experiments showed a major 130 kDa band when using DSS at either a 0.2 or a 0.4 mM concentration (the lower DSS concentration gave somewhat cleaner results by reducing cross-linking in the minor 90 and 33 kDa membrane components). A 135 kDa ANP-R t receptor cDNA has been cloned for rat brain (Chinkers et al., 1989). Similar 130 kDa receptors have been identified in astrocyte-predominant cultures from rat fetal diencephalon (Levin et al., 1990). Our human neuronal cell line indicates that similar receptors can undoubtedly be expressed outside gila. Their Mr is definitely higher than that of 115 kDa predicted for ANP-R 3 receptors also coupled to cyclic GMP production, that have been cloned from human brain cDNA and which express preference for BNP (Chang et al., 1989). In addition, we observed no 66 kDa band corresponding to ANP-R 2 receptors under reducing conditions (ANP-R 2 receptors are dimers of two identical 66 kDa subunits linked by a disulfide bridge; Leitman et al., 1988). Our data on human neuroblastoma membranes were in line with the presence of ANP-R t receptors (K o of 100 pM) tested for ligand binding and cyclic GMP elevation in intact mouse neuroblastoma N4TG1 cells (Pandey et al., 1987) and in various brain regions including the choroid plexus, circumventricular areas implicated in cardiovascular and fluid homeostasis (Brown and Czarnecki, 1990), thalamus, cerebellum (Quirion et al., 1986) and olfactory bulb (Agui et al., 1989). On the

87 other hand, ANP-R2-type receptors have been characterized in pituitary and arachnoid mater (Brown and Czarnecki, 1990). The presence of the disulfide bridge in A N P was indispensable for b i n d i n g to NB-OK-1 R~ receptors as rat ANP-(111-126), rat ANP-(99-109) and rat (des Cysl°S,Cys121) ANP-(104-126), all ring deleted, were unable to recognize these receptors. The C-terminal extremity but not the N-terminal extremity was also essential. Indeed, h u m a n ANP-(102-126), truncated only at the N-terminal extremity, showed almost the same affinity (55 pM) as h u m a n ANP-(99-126) (30 pM) while rat ANP-(103-123) and rat C-ANP-(102-121), truncated at both extremities, showed no b i n d i n g affinity. Some of the amino acids under the 105-121 disulfide bridge were less important for binding. Indeed BNP, which besides lacking three amino acid residues at the N-terminus also shows four changes in the ring structure (Sudoh et al., 1988), was only 10 times less potent than rat ANP-(99126). BNP has, however, one supplementary arginine in its C-terminal part that might contribute to b i n d i n g affinity. Similar conclusions have been reached in other tissues and cell lines (Bellemann and Neuser, 1988; Olins et al., 1988; Neuser et al., 1988). Dithiothreitol (DTT), often used in guanylate cyclase assays in order to protect thiol groups close to the catalytic center ( W a l d m a n and Murad, 1987), shifted the A N P dose-response curve of the enzyme rightward by a factor of 30. It is probable that D T T partly reduced the disulfide bridge of ANP, which indicates that A N P effects on guanylate cyclase should not be measured in the presence of D T T as often is the case. The expression of A N P receptors increased in neuroblastoma cells treated with I m M carbamylcholine or 1 m M D B c A M P by 36% and 125%, respectively, after 24 h. These data were well correlated with corresponding increases in Vma~ of guanylate cyclase exposed to A N P (35% and 122% in membranes from cells exposed to carbamylcholine and DBcAMP, respectively) (fig. 7). There was no alteration in the ECs0 of the enzyme. This increased n u m b e r of A N P receptors was already significant after an 8 h exposure to both agents. However, when cells were treated for 8 h with carbamylcholine or D B c A M P in the added presence of cycloheximide, the n u m b e r of receptors did not increase, indicating that protein synthesis was involved in receptor induction. Interestingly, we observed that the n u m b e r of A N P receptors in neuroblastoma cells treated with rat ANP(99-126) for times up to 24 h did not change, contrasting with the reported down-regulation of about 50% induced by similar doses of A N P in vascular smooth muscle cells after 24 h (Hirata et al., 1985a,b and 1986; Roubert et al., 1987). This discrepancy might reflect rapid degradation of A N P in neuroblastoma cells a n d / o r preferential down-regulation of A N P - R 2 receptors in vascular smooth muscle cells.

It is tempting to conclude that our results, showing a variable expression of functional ANP-R~ receptors in a h u m a n neuronal cell line, support a regulatory role for A N P in central brain areas endowed with A N P - R 1 receptors.

Acknowledgements Aided by Grant 3.4504.85 from the Fund for Medical Scientific Research (Belgium) and Grant 87/92408 from a "Concerted Research Action" from the Ministry of ScientificPolitics(Belgium). We thank Dr. Gillian M. Olins (Searle, St Louis, MO) for the generous gift of [Des Cysl°5,Cys121]ANP-(104-126).Christine Delporte is a recipient of a predoctoral fellowship from IRSIA (Belgium). The authors thank N. Peuchot and M. Sti~venartfor their secretanal help.

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