Functional analysis of the glucose response element of the rat glucagon receptor gene in insulin-producing INS-1 cells.

Glucose stimulates the transcription of the glucagon receptor gene in hepatocytes and in pancreatic beta cells. We recently identified a glucose response element in the immediate upstream non-coding region of the rat glucagon receptor gene. We previously showed that this DNA element is centered on a palindromic sequence of 19 nucleotides (termed as G box), containing two E boxes separated by three nucleotides. In the present study, we further characterized the DNA sequence requirements for the glucose induced expression of the rat glucagon receptor gene. Transfection study realized in the insulin-producing INS-1 cells revealed that a fragment of 49 nucleotides, centered on the G box, bears all the features required for the glucose activation. Mutations performed in the 5'-E box totally abolished the glucose responsiveness, whereas mutations or deletion of the 3'-E box only had a limited effect. Deletions performed upstream from the G box revealed that an accessory factor binding site, located in the region just upstream from the G box, is required for full stimulation by glucose. Finally, by using G box based probes in gel shift experiments, we demonstrated that USF1/USF2 transcription factors are part of the proteinic complex that binds to the glucose response element of the rat glucagon receptor gene promoter. In conclusion, in contrast to many other glucose regulated genes, only the 5'-E box appears to be a crucial DNA element for the glucose transcriptional effect. However, an accessory factor binding site located in the region just upstream from the G box is required for a complete stimulation by glucose.

Inhibitory effects of ATP and other nucleotides on atrial natriuretic peptide (ANP) binding to R1-type ANP receptors in human neuroblastoma NB-OK-1 cell membranes.

ATP dose-dependently inhibited rat 125I-ANP-(99-126) binding to membranes from the human neuroblastoma cell line NB-OK-1 by increasing the KD value for the hormone without altering the Bmax value. After a 20 min preincubation with 37.5 pM 125I-ANP-(99-126) and 0.5 mM ATP, followed by the addition of 0.3 microM unlabelled ANP-(99-126), the proportion of rapidly dissociating receptors was 4-times higher than in the absence of ATP. The other nucleotides ADP, AMP, AMP-PNP, ATP gamma S, GTP, GDP, GMP, GMP-PNP and GTP gamma S were also inhibitory but with a lower potency and/or efficacy. Binding equilibrium data were satisfactorily simulated by a computer program based on partially competitive binding of ANP-(99-126) and the nucleotides, and this, together with the data on dissociation kinetics, strongly suggests that several nucleotides, when added at concentrations up to 1 mM, form a ternary ANP-receptor-nucleotide complex.

Pharmacology of hexahydro-difenidol, hexahydro-sila-difenidol and related selective muscarinic antagonists.

A series of hexahydro-difenidol (HHD) and hexahydro-sila-difenidol (HHSiD) analogues modified in the amino group, the phenyl ring and in the alkylene chain were investigated for their binding and functional properties at muscarinic M1, M2 and M3 receptors. Novel muscarinic receptor antagonists were obtained which exhibited different receptor selectivity profiles from the parent compounds HHD and HHSiD (M1 congruent to M3 greater than M2), e.g. HHD and HHSiD methiodides, M1 greater than M2 congruent to M3; p-fluoro-HHSiD, M3 greater than M1 greater than M2; trans-hexbutenol, M1 greater than M3 greater than M2; and (s)-p-fluoro-hexbutinol, M3 greater than M2 congruent to M1. Stereoselectivity ratios [(R)/(S)] for the enantiomers of HHD, hexbutinol and p-fluoro-hexbutinol were highest at M1, intermediate at M3 and lowest at M2 receptors.

Stereoselectivity of the interaction of muscarinic antagonists with their receptors.

The stereoselectivity of the interaction with muscarinic receptors of enantiomers of a series of chiral antagonists is receptor subtype dependent. There is no overall relationship between stereoselectivity and receptor affinity. Depending on the antagonist studied, receptor stereoselectivity may indeed reflect: (1) the weakening or loss of a single interaction involving one of the four groups bound to the asymmetric carbon; (2) steric hindrance preventing optimum interaction of the low affinity steroisomer with the receptor; and/or (3) the inversion of the relative positions of two moieties of the ligand with similar structural and electronic properties i.e. comparable affinities for the two corresponding subsites in the receptor.

Sequencing of eleven introns in genomic DNA encoding rat glucagon receptor and multiple alternative splicing of its mRNA.

We used the PCR (polymerase chain reaction) to amplify fragments of glucagon receptor DNA from genomic DNA. Sequencing of the subcloned fragments demonstrated that genomic DNA encoding the glucagon receptor spans over 12 exons interrupted by 11 introns. The introns were located mainly at the 5' end and in the core domain of the glucagon receptor CDS totalling 23 kb. Intron positions were similar to the positions of introns in growth hormone-releasing hormone receptor and parathyroid hormone receptor, two receptors belonging to the same receptor family as the glucagon receptor. This high number of introns might be the cause of the mRNA polymorphism observed at the 5' end: when PCR was performed on cDNA using primers amplifying the central or 3' end cDNA fragments, a single band corresponding to the cloned cDNA was observed. In contrast, if primers amplifying cDNA fragments corresponding to nucleotides -8 to 680 of CDS were used, cDNA fragments of approximately 500 bp, 600 bp, 700 bp, 800 bp and 900 bp were specifically and reproducibly amplified. Sequencing of these fragments showed either incomplete intron removal or splicing at alternative positions. Two of these sequenced variants were translatable in putative glucagon receptor variants: (1) unsplicing of intron III (81 bp) gave an additional 27 amino acid sequence after Lys91 in the N-terminal domain of the receptor. In the liver, where the normal CDS represented about one third of the mRNA molecules, this mRNA variant represented 18% of total mRNA forms; (2) a 21 bp deletion in exon V giving rise to a putative deletion of 7 amino acids in glucagon receptor (delta 64-84 CDS) was also relatively abundant in the liver (10%). The observed polymorphism of the glucagon receptor mRNA may contribute to the regulation of glucagon receptor expression and perhaps to the heterogeneity of these receptors.

80% of muscarinic receptors expressed by the NB-OK 1 human neuroblastoma cell line show high affinity for pirenzepine and are comparable to rat hippocampus M1 receptors.

The NB-OK 1 human neuroblastoma cell line expressed muscarinic cholinergic receptors that could be labeled with N-[3H]methylscopolamine (a nonselective antagonist). 80% of these receptors showed high affinity for pirenzepine, i.e. belonged to the M 1 subtype found in neuronal tissues. Their binding properties were identical to those of rat hippocampus M 1 receptors, and differed from those of rat pancreas and heart muscarinic receptors. The remaining (20%) muscarinic receptors showed low affinity for pirenzepine and AF-DX 116, being therefore of an M2 beta (or B) subtype, and were similar to rat pancreatic receptors.

Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB-OK 1.

125I-[Tyr2]scyllatoxin allowed to label a single class of high-affinity receptors in membranes from the human neuroblastoma cell line NB-OK 1. The Kd of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the Bmax was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5-fold after a 24-h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide.

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts.

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.

Binding properties of nine 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP) analogues to M1, M2, M3 and putative M4 muscarinic receptor subtypes.

1. We compared the binding properties of 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) and nine analogues of this compound on muscarinic receptors of human neuroblastoma NB-OK1 cells (M1 subtype), rat heart (M2 subtype), rat pancreas (M3 subtype) and to the putative M4 subtype in striatum. 2. The requirements for high affinity binding were somewhat different for the four receptor subtypes. In general, the requirements of M3 receptors were more stringent than for M1, M2 or putative M4 receptors. 3. The abilities of the compounds to discriminate muscarinic receptor subtypes were not correlated with their affinities at any subtype. 4. The temperature-dependence of binding of 4-DAMP analogues to M2 receptors varied with the drug structure. In particular, the increased affinity of the alpha-methyl derivative of 4-DAMP could be ascribed to van der Waals interactions. 5. The affinities of most 4-DAMP analogues for M2 and M3 receptors were similar to their pharmacological potencies on atrial and ileum preparations, respectively. 6. At concentrations above 1 microM, all 4-DAMP analogues as well as atropine, reduced the [3H]-N-methyl scopolamine ([3H]-NMS) dissociation rate from cardiac muscarinic receptors, with no obvious structure-activity relationship.

Stereoselective recognition of the enantiomers of phenglutarimide and of six related compounds by four muscarinic receptor subtypes.

1. We have compared the binding properties of the enantiomers of phenglutarimide (1) and of six related compounds to M1 receptors in NB-OK-1 cells, M2 receptors in rat heart, M3 receptors in rat pancreas and the M4 receptors of rat striatum, with their functional (antimuscarinic) properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2) and guinea-pig ileum (M3) receptors. The binding properties of the enantiomers of three of the compounds were also measured on cloned human m1-m4 receptors expressed by CHO cells, using [3H]-N-methylscopolamine ([3H]-NMS) as radioligand. 2. The high affinity enantiomers behaved as competitive antagonists in binding and pharmacological studies. (S)-phenglutarimide (pKi-M1 = 9.0/9.3) and (R)-thienglutarimide (pKi-M1 = 8.6/9.2) recognized selectively the native M1 > M4 > M3 > M2 receptors in tissues as well as the respective cloned receptors. 3. The pA2 values at the inhibitory heteroreceptors in the rabbit vas deferens, and at the guinea-pig atria and ileum for the seven more potent enantiomers were compatible with the previous classification of these receptors as M1/M4-like, M2 and M3, respectively. 4. Replacement of the phenyl by a thienyl ring or of the diethylamino by a piperidino group in the phenglutarimide molecule did not affect markedly the potencies of the high affinity enantiomer. In contrast, replacement of the phenyl by a cyclohexyl ring decreased 20 fold the active enantiomers potency. Methylation of the piperidine-2,6-dione nitrogen also reduced markedly the eutomers' affinities, more on the M1 than on the other subtypes. 5. The selectivity profiles (recognition of four receptor subtypes) of six of the seven less active enantiomers were different from the corresponding more active enantiomers selectivity profiles, suggesting that the preparations used in this study were pure. However, we cannot not exclude the hypothesis that the batch of (S)-thienglutarimide used in this study was contaminated by less than 0.02% of the eutomer. 6. In contrast with the eutomer binding site, replacement of the phenyl ring by a thienyl or cyclohexyl ring did not affect binding of the low affinity enantiomers to the muscarinic receptor or the [3H]-NMS-receptor complex. The replacement of the diethylamino group by a piperidine ring, and N-methylation of the piperidine-2,6 dione moiety increased slightly these enantiomers' potencies. 7. The muscarinic receptors were extremely stereoselective, and had up to 20000 fold lower affinity for the less active enantiomers. However, the stereochemical requirements of the muscarinic receptor subtypes were different for the enantiomers of compounds 1-7, being most stringent at M1 receptors. 8. The weaker enantiomers behaved as competitive antagonists in pharmacological studies, at least in the concentration-range investigated.

Binding and functional properties of hexocyclium and sila-hexocyclium derivatives to muscarinic receptor subtypes.

1. We have compared the binding properties of several hexocyclium and sila-hexocyclium derivatives to muscarinic M1 receptors (in rat brain, human neuroblastoma (NB-OK 1) cells and calf superior cervical ganglia), rat heart M2 receptors, rat pancreas M3 receptors and M4 receptors in rat striatum, with their functional antimuscarinic properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2), and guinea-pig ileum (M3) muscarinic receptors. 2. Sila-substitution (C/Si exchange) of hexocyclium (-->sila-hexocyclium) and demethyl-hexocyclium (-->demethyl-sila-hexocyclium) did not significantly affect their affinities for muscarinic receptors. By contrast, sila-substitution of o-methoxy-hexocyclium increased its affinity 2 to 3 fold for all the muscarinic receptor subtypes studied. 3. The p-fluoro- and p-chloro-derivatives of sila-hexocyclium had lower affinities than the parent compound at the four receptor subtypes, in binding and pharmacological studies. 4. In binding studies, o-methoxy-sila-hexocyclium (M1 = M4 > or = M3 > or = M2) had a much lower affinity than sila-hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila-hexocyclium (M1 = M3 > M4 > M2). This is in marked contrast with the very clear selectivity of o-methoxy-sila-hexocyclium for the prejunctional M1/M4-like heteroreceptors in rabbit vas deferens. 5. The tertiary amines demethyl-hexocyclium, demethyl-sila-hexocyclium and demethyl-o-methoxy-sila-hexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives.

Binding and functional properties of antimuscarinics of the hexocyclium/sila-hexocyclium and hexahydro-diphenidol/hexahydro-sila-diphenidol type to muscarinic receptor subtypes.

1. In an attempt to assess the structural requirements for the muscarinic receptor selectivity of hexahydro-diphenidol (hexahydro-difenidol) and hexahydro-sila-diphenidol (hexahydro-sila-difenidol), a series of structurally related C/Si pairs were investigated, along with atropine, pirenzepine and methoctramine, for their binding affinities in NB-OK 1 cells as well as in rat heart and pancreas. 2. The action of these antagonists at muscarinic receptors mediating negative inotropic responses in guinea-pig atria and ileal contractions has also been assessed. 3. Antagonist binding data indicated that NB-OK 1 cells (M1 type) as well as rat heart (cardiac type) and pancreas (glandular/smooth muscle type) possess different muscarinic receptor subtypes. 4. A highly significant correlation was found between the binding affinities of the antagonists to muscarinic receptors in rat heart and pancreas, respectively, and the affinities to muscarinic receptors in guinea-pig atria and ileum. This implies that the muscarinic binding sites in rat heart and the receptors in guinea-pig atria are essentially similar, but different from those in pancreas and ileum. 5. The antimuscarinic potency of hexahydro-diphenidol and hexahydro-sila-diphenidol at the three subtypes was influenced differently by structural modifications (e.g. quaternization). Different selectivity profiles for the antagonists were obtained, which makes these compounds useful tools to investigate further muscarinic receptor heterogeneity. Indeed, the tertiary analogues hexahydro-diphenidol (HHD) and hexahydro-sila-diphenidol (HHSiD) had an M1 = glandular/smooth muscle greater than cardiac selectivity profile, whereas the quaternary analogues HHD methiodide and HHSiD methiodide were M1 preferring (M1 greater than glandular/smooth muscle, cardiac).

Thermodynamics of antagonist binding to rat muscarinic M2 receptors: antimuscarinics of the pridinol, sila-pridinol, diphenidol and sila-diphenidol type.

1. We studied the effect of temperature on the binding to rat heart M2 muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila-pridinol and diphenidol/sila-diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)- and (S)-procyclidine, (R)- and (S)-trihexyphenidyl, (R)- and (S)-tricyclamol, (R)- and (S)-trihexyphenidyl methiodide, (R)- and (S)-hexahydro-diphenidol and (R)- and (S)-hexbutinol]. Binding affinities were determined in competition experiments using [3H]-N-methyl-scopolamine chloride as radioligand. The reference drugs were scopolamine and N-methyl-scopolamine bromide. 2. The affinity of the antagonists either increased or decreased with temperature. van't Hoff plots were linear in the 278-310 degrees K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to -29 kJ mol-1) to large positive values (up to +30 kJ mol-1). 3. (R)-configurated drugs had a 10 to 100 fold greater affinity for M2 receptors than the corresponding (S)-enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed. 4. When silanols (R3SiOH) were compared to carbinols (R3COH), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds (R4Ge) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes. 5. Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change of drug binding. 6. Replacement of a pyrrolidino by a piperidino group and increasing the length of the alkylene chain bridging the amino group and the central carbon or silicon atom were associated with either an increase or a decrease of entropy and enthalpy changes of drug binding. However, there was no clear correlation between these structural variations and the thermodynamic effects. 7. Taken together, these results suggest that hydrogen bond-forming OH groups and, to a lesser extent, polarizable phenyl groups contribute significantly to the thermodynamics of interactions between these classes of muscarinic antagonists and M2 muscarinic receptors.

Inactivation of brain cortex muscarinic receptors by 4-diphenylacetoxy-1-(2-chloroethyl) piperidine mustard.

We demonstrated in this study that 4-DAMP [4-diphenylacetoxy-1-(2- chloroethyl) piperidine] mustard, which cyclizes to the aziridinium ion, behaved as a non-selective, non-competitive inhibitor of muscarinic receptors in rat brain cortex. It inactivated to the same extent the M1, M2 and M4 muscarinic receptors present in this tissue, as well as receptors accessible or not accessible to quaternary antimuscarinic drugs. Under mild incubation conditions, the muscarinic receptors in a state with super high affinity for agonists (SH receptors) were less affected by preactivated 4-DAMP mustard than the receptors in the states with lower affinity for agonists (H and L receptors).

Relative quantitative analysis of glucagon receptor mRNA in rat tissues.

Total RNA prepared from nine rat tissues were analyzed for their content in glucagon receptor mRNA by two independent hybridization approaches: (1) simple dot blot analysis using labelled oligodeoxynucleotide; (2) highly specific RNase protection assay using labelled antisense RNA. Hybridization signal was quantified by laser densitometric scanning of autoradiographies. Results were expressed for each method relative to the liver content (100%) for either a constant amount of total RNA or for a constant amount of beta-actin mRNA. We obtained similar relative values of glucagon receptor mRNA per constant amount of total RNA by the two hybridization methods: in liver (100 and 100), in kidney (38 and 34), and in heart (12 and 11). The glucagon receptor mRNA was overestimated by the less specific dot assays, in adrenal glands (21 versus 10) and in adipose tissues (24 versus 5). In the stomach, brain, duodenum and lung, the signal was equal to or below the reliable quantification limit. Reverse transcription and polymerase chain reaction (RT-PCR) of glucagon receptor mRNA with limited cycle number were performed using two sets of primers: the first set amplified a single band at the 3' coding end, and the second, 3-6 bands at the 5' coding end, revealing tissue-specific polymorphism. RT-PCR data confirmed the presence of glucagon receptor mRNA in liver, kidney, heart, adrenal glands and adipose tissue, and allowed the detection of a very low amount of glucagon receptor mRNA in the stomach, the duodenum and brain but not in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutational analysis of the glucose regulatory element in the promoter of the glucagon receptor gene.

Recently, we identified a glucose regulatory element in the promoter of the rat glucagon receptor gene. The effect of glucose is centered on a highly palindromic sequence of 19 nucleotides that we called the G box (Portois et al., 1999, J. Biol. Chem. 274: 8181-8190). This sequence contains two E boxes. Recently, we investigated the role of each individual E box, as well as the contribution of the sequences located upstream and downstream from this G box. (1) Mutation of nucleotides "CA" to "GT" in the first E box (position -543 to -542) suppressed the activation of the CAT reporter gene by glucose. In contrast, mutation of the nucleotides "CA" to "GT" in the second E box (position -534 to -533) had no effect on this glucose activation. (2) Deletion of a sequence upstream from the G box (nucleotides -579 to -555) suppressed the activation by glucose, whereas deletion of a sequence located downstream from the G box (nucleotides -501 to -443) had no effect on this parameter. (3) Subcloning of a small promoter fragment of only 49 nucleotides (-560 to -512) into the pCat5 plasmid conferred to transfected cells sensitivity to glucose in terms of CAT activity. Consequently, all transactivation factors required for this glucose effect must act via this short gene fragment.

VIP and related peptides induce rapid homologous desensitization in the human lymphoma SUP T1 cell line.

Incubation of human SUP T1 lymphoblasts with VIP, helodermin and related peptides induced homologous desensitization within 5 min as indicated by: 1) a secondary decrease in cellular cyclic AMP levels, even in the presence of phosphodiesterase inhibitors, 2) a reduced capacity of cells to bind [125I]helodermin, 3) decreased helodermin stimulation of adenylate cyclase activity in membranes, and 4) unaffected NaF- and Gpp[NH]p-stimulated adenylate cyclase activities. The desensitizing ability of all peptides correlated with their efficacy to occupy cell receptors, except for [D-Phe2]VIP, a partial VIP agonist with low intrinsic activity, that did not desensitize.

Molecular cloning and in vitro properties of the recombinant rabbit secretin receptor.

The rabbit secretion receptor cDNA was cloned from rabbit pancreas using combined polymerase chain reaction (PCR)/rapid amplification of cDNA ends (PCR/RACE) approaches. The rabbit cDNA encoded 445 amino acids and had 80 and 85% homology with rat- and human receptor, respectively, in terms of nucleic and amino acid sequences. Several regions where the rabbit receptor sequence diverged from the rat/human receptor sequences were observed in the putative extracellular domains of the receptor. A cDNA coding for a similar sequence with a 76 bp deletion after the 5th transmembrane domain was also found; it probably encoded an inactive protein. The whole rabbit secretin receptor cDNA was subcloned in expression vector pCR3.1, then stably and transiently transfected in Chinese hamster ovary (CHO) cells. The pharmacological properties of the rat and rabbit secretin receptor studies were compared by radiolabeled secretin binding, binding inhibition, and adenylate cyclase activation (using secretin analogs and fragments). Porcine secretin was equipotent with rabbit secretin on the rabbit secretin receptor, but fivefold more potent than rabbit secretin on the rat receptor. This was due to the serine-->arginine residue replacement, in position 16 of rabbit secretin. Amino terminal modified secretin analogs (secretin (2-27), [E3]secretin, [N3]secretin) and VIP were less potent than secretin on both secretin receptors, but more potent on the rabbit than on the rat receptor. The carboxy-terminally truncated fragment (1-26) had the same reduced potency on rat and rabbit receptors. Thus, the rabbit secretin receptor had original properties, different from those of the rat receptor.

Atrial natriuretic peptide binds to ANP-R1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond.

ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.

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

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R1 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) greater than human ANP-(99-126) greater than human ANP-(102-126) greater than porcine BNP (brain natriuretic peptide). The enzyme required free Mn2+ in addition to the Mn-GTP substrate (Km 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-R1 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).