RESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN , Glucosa/genética , Insulina/metabolismo , Receptores de Glucagón/genética , Animales , Línea Celular , Elementos E-Box , Eliminación de Gen , Glucosa/farmacología , Ratas , Receptores de Glucagón/efectos de los fármacos , Elementos de Respuesta , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , Factores Estimuladores hacia 5'RESUMEN
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.
Asunto(s)
Glucosa/farmacología , Mutación , Regiones Promotoras Genéticas , Receptores de Glucagón/genética , Animales , Secuencia de Bases , Línea Celular , ADN/genética , Análisis Mutacional de ADN , Cartilla de ADN/genética , Genes Reguladores/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , TransfecciónRESUMEN
We cloned the 5' upstream region of the rat glucagon receptor gene, demonstrating that the 5' noncoding domain of the glucagon receptor mRNA contained two untranslated exons of 131 and 166 nucleotides (nt), respectively, separated by two introns of 0.6 and 3.2 kilobase pairs. We also observed an alternative splicing involving the 166-base pair exon. Cloning of up to 2 kilobase pairs of the newly identified genomic domain and transfection of various constructs driving a reporter gene, in pancreatic islet cell line INS-1, uncovered a strong glucose regulation of the promoter activity of plasmids containing up to nucleotide -868, or more, upstream from the transcriptional start point. This promoter activity displayed threshold-like behavior, with low activity of the promoter below 5 mM glucose, and maximal activation as of 10 mM glucose. This glucose regulation was mapped to a highly palindromic 19-nucleotide region between nt -545 and -527. Indeed, deletion or mutation of this sequence abolished the glucose regulation. This domain contained two palindromic "E-boxes" CACGTG and CAGCTG separated by 3 nt, a feature similar to the "L4 box" found in the pyruvate kinase L gene promoter. This is the first description of a G protein-coupled receptor gene promoter regulated by glucose.
Asunto(s)
ADN/metabolismo , Glucosa/metabolismo , Regiones Promotoras Genéticas , Receptores de Glucagón/genética , Animales , Secuencia de Bases , Southern Blotting , Cartilla de ADN/metabolismo , Biblioteca de Genes , Datos de Secuencia Molecular , Técnica del ADN Polimorfo Amplificado Aleatorio , Ratas , TransfecciónRESUMEN
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.
Asunto(s)
Receptores de la Hormona Gastrointestinal/química , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , Humanos , Datos de Secuencia Molecular , Unión Proteica , Conejos , Ratas , Receptores Acoplados a Proteínas G , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secretina/análogos & derivados , Secretina/metabolismoRESUMEN
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.
Asunto(s)
Glutetimida/análogos & derivados , Parasimpatolíticos/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Células CHO , Cricetinae , Femenino , Glutetimida/química , Glutetimida/metabolismo , Cobayas , Humanos , Íleon/efectos de los fármacos , Íleon/metabolismo , Técnicas In Vitro , Masculino , Membranas/metabolismo , Antagonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacología , Miocardio/metabolismo , Parasimpatolíticos/química , Conejos , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores Muscarínicos/efectos de los fármacos , Estereoisomerismo , Relación Estructura-Actividad , Conducto Deferente/metabolismoRESUMEN
We have cloned and sequenced a cDNA isolated from a human SUP-T1 lymphoblast cell line library. It encoded a 457 amino acids protein having 87% identity with the rat PACAP type II, VIP2 receptor. Chinese hamster ovary (CHO) cells stably transfected with cloned cDNA expressed a specific binding of 125I[Acetyl-His1]PACAP-27. This binding was inhibited by GTP, and by the peptides helodermin, VIP, PACAP-27 and PACAP-38 that also stimulated adenylate cyclase activity. The order of potency was PACAP-38 > VIP > or = helodermin > or = PACAP-27. Comparison of the results in two cell lines expressing different receptor densities suggested that helodermin and PACAP-38 had a higher intrinsic activity than VIP and PACAP-27.
Asunto(s)
Linfocitos/metabolismo , Receptores de Péptido Intestinal Vasoactivo/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , Cartilla de ADN/genética , ADN Complementario/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Péptidos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Homología de Secuencia de Aminoácido , Transfección , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
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)
Asunto(s)
Riñón/química , Hígado/química , ARN Mensajero/análisis , Receptores de Glucagón/genética , Actinas/análisis , Actinas/genética , Tejido Adiposo/química , Tejido Adiposo/metabolismo , Tejido Adiposo/ultraestructura , Glándulas Suprarrenales/química , Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/ultraestructura , Animales , Autorradiografía , Northern Blotting , Química Encefálica , Densitometría , Duodeno/química , Duodeno/metabolismo , Duodeno/ultraestructura , Mucosa Gástrica/metabolismo , Riñón/metabolismo , Riñón/ultraestructura , Hígado/metabolismo , Hígado/ultraestructura , Pulmón/química , Pulmón/metabolismo , Pulmón/ultraestructura , Miocardio/química , Miocardio/metabolismo , Miocardio/ultraestructura , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , ARN Mensajero/genética , Ratas , Receptores de Glucagón/análisis , Receptores de Glucagón/metabolismo , Estómago/química , Estómago/ultraestructuraRESUMEN
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.
Asunto(s)
Empalme Alternativo , Receptores de Glucagón/genética , Animales , Clonación Molecular , Variación Genética , Genoma , Intrones/genética , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Parasimpatolíticos/farmacología , Piperazinas/farmacología , Piperazinas/farmacocinética , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Animales , Bovinos , Femenino , Cobayas , Humanos , Técnicas In Vitro , Masculino , N-Metilescopolamina , Parasimpatolíticos/farmacocinética , Pirenzepina/farmacología , Conejos , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Derivados de Escopolamina/farmacología , Relación Estructura-Actividad , Células Tumorales Cultivadas , Conducto Deferente/efectos de los fármacosRESUMEN
The rat pancreatic acinar cell line AR 4-2J is endowed with numerous PACAP type I receptors (PACAPR1). The cDNA of this receptor was PCR amplified at low stringency using two degenerate primers based on conserved sequences in the TM2 and TM6 segments of secretin, parathormone and calcitonin receptors. One additional amplified band of 574 bp possessed an original 84 bp insert. This fragment, when used as probe for Northern blot analysis, revealed a high M(r) (about 7.5 kb) transcript in AR 4-2J cells and also in rat brain. Screening a lambda Uni-Zap bacteriophage library of AR 4-2J cell cDNA yielded one hybridizing clone with an ORF of 1254 bp. The translated 418 amino acid peptide showed 206 identities with rat VIP receptors and 176 identities with secretin receptors. This sequence homology, together with the mRNA distribution, and the expression study of a similar cDNA published very recently (8) indicated that we had cloned PACAPR1 except for its 77 N-terminal amino acids. Its putative I3 loop contained a unique additional 28 amino acid sequence (with four hemi-cystines and several serine, threonine and basic residues). Using RT-PCR we then demonstrated the coexistence of a second form of mRNA, without this added insert, in DNAse-pretreated RNAs from both AR 4-2J cells and normal rat brain. This indicated that common alternative splicing provokes the diversification of PACAP type I receptors into PACAPR1A (unspliced) and PACAPR1B (spliced) variants.
Asunto(s)
Empalme Alternativo , Encéfalo/metabolismo , Neuropéptidos/metabolismo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores de la Hormona Hipofisaria , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Clonación Molecular , Secuencia Conservada , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Páncreas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Receptores de Superficie Celular/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Parasimpatolíticos/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Corazón/efectos de los fármacos , Antagonistas de los Receptores Histamínicos H1/farmacología , Técnicas In Vitro , Masculino , N-Metilescopolamina , Parasimpatolíticos/farmacología , Piperidinas/farmacología , Ratas , Ratas Endogámicas WKY , Receptores Muscarínicos/efectos de los fármacos , Derivados de Escopolamina/metabolismo , Estereoisomerismo , TermodinámicaRESUMEN
We constructed a full open reading frame (ORF) for the rat hepatic glucagon receptor using two non functional pCDM8 clones of a putative glucagon receptor. Clone I was complete but contained, in addition, two small introns interrupting the frame, while clone II lacked 104 bp at the 5' end. We isolated Hind III/Dra III and BamH I/Not I fragments from clone I and a Dra III/BamH I fragment from clone II then ligated the three fragments with pBluescript SK(+) digested with Hind III/Not I. Following this 4 fragment ligation procedure, we obtained one correct insert in a clone we subcloned in the Hind III/Not I sites of pCDM8. This plasmid was used to transiently transfect COSGs1 cells with the lipofectin method. In COSGs1 membranes transfected with this correct plasmid (but not with a truncated plasmid) [125I]iodoglucagon binding was selectively displaced with glucagon (IC50 = 4 nM) and adenylate cyclase was stimulated with glucagon (K(act) = 10 nM) while related peptides were inefficient at 1 microM. This demonstrated that the receptor previously described is indeed the rat hepatic glucagon receptor.
Asunto(s)
Glucagón/metabolismo , Hígado/metabolismo , Receptores de la Hormona Gastrointestinal/genética , Animales , Línea Celular , Clonación Molecular , ADN , Haplorrinos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Ratas , Receptores de Glucagón , TransfecciónRESUMEN
A cDNA fragment was PCR amplified at low-stringency from rat DNA, using two degenerated primers corresponding to consensus sequences in the second and third transmembrane segments of members of the secretin receptor family. A synthetic oligodeoxynucleotide based on the 3 end of this fragment, when used in Northern-blot analysis, hybridized to a broad 1.5-3.0 kb band in rat liver. It was then used as a probe for screening a cDNA library generated from size-fractionated rat liver poly(A)+ RNA. Overlapping alignment of sequences from 3 clones identified a 1455 residue open reading frame encoding a 485 amino acid peptide showing 7 putative transmembrane segments and 42% identity with the glucagon-like peptide 1 (GLP-1) receptor. In addition, four small (85-300 bp) introns were variously represented in the isolated clones.
Asunto(s)
Intrones , Hígado/metabolismo , Receptores de Glucagón , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN , Receptor del Péptido 1 Similar al Glucagón , Datos de Secuencia Molecular , Ratas , Receptores de Superficie Celular/genética , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Animales , Factor Natriurético Atrial/farmacología , Supervivencia Celular , Pollos , Cromatografía Líquida de Alta Presión , GMP Cíclico/análisis , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Radioisótopos de Yodo , L-Lactato Deshidrogenasa/análisis , Metaloendopeptidasas/metabolismo , Neuroblastoma/metabolismo , Fragmentos de Péptidos/metabolismo , Ratas , Porcinos , Células Tumorales CultivadasRESUMEN
We investigated the binding properties of the (R)- and (S)-enantiomers of the muscarinic antagonists trihexyphenidyl, procyclidine, hexahydro-difenidol, p-fluoro-hexahydro-difenidol, hexbutinol, p-fluoro-hexbutinol, and their corresponding methiodides at muscarinic M1, M2, M3 and M4 receptor subtypes. In addition, binding properties of the (R)- and (S)-enantiomers of oxyphencyclimine were studied. The (R)- enantiomers (eutomers) of all the compounds had a greater affinity than the (S)-isomers for the four muscarinic receptor subtypes. The binding patterns of the (R)- and (S)-enantiomers were generally different. We did not observe any general correlation between the potency of the high-affinity enantiomer and the affinity ratio (eudismic ratio) of the two enantiomers. The results are discussed in terms of a 'four subsites' binding model.
Asunto(s)
Alquinos/farmacología , Piperidinas/farmacología , Prociclidina/farmacología , Pirimidinas/farmacología , Receptores Muscarínicos/efectos de los fármacos , Animales , Cuerpo Estriado/efectos de los fármacos , Corazón/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , N-Metilescopolamina , Neuroblastoma , Páncreas/efectos de los fármacos , Parasimpatolíticos/farmacología , Ratas , Ratas Endogámicas , Derivados de Escopolamina/farmacología , Estereoisomerismo , TritioRESUMEN
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).
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Ácidos Difenilacéticos/farmacología , Piperidinas/farmacología , Receptores Muscarínicos/efectos de los fármacos , Animales , Carbacol/metabolismo , Corteza Cerebral/metabolismo , Antagonistas Muscarínicos , Miocardio/metabolismo , Ratas , Ratas EndogámicasRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/farmacología , Factor Natriurético Atrial/metabolismo , Neuroblastoma/metabolismo , Nucleótidos/farmacología , Fragmentos de Péptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Adenosina Monofosfato/farmacología , Animales , Unión Competitiva , Membrana Celular/metabolismo , Humanos , Cinética , Ratas , Receptores del Factor Natriurético Atrial , Receptores de Superficie Celular/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Piperidinas/metabolismo , Receptores Muscarínicos/metabolismo , Derivados de Escopolamina/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Cuerpo Estriado/metabolismo , Técnicas In Vitro , Masculino , Miocardio/metabolismo , N-Metilescopolamina , Neuroblastoma/metabolismo , Páncreas/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Endogámicas , Relación Estructura-Actividad , Temperatura , Células Tumorales CultivadasRESUMEN
We analyzed the competition kinetics of quinuclidinyl benzilate (QNB) and QNB methiodide enantiomers on human NB-OK1 neuroblastoma (M1), rat cardiac (M2), and rat pancreas (M3) muscarinic binding sites. The association rate constants of the four drugs depended on the receptor subtype studied and were lower with pancreas (M3) (1-9 x 10(5) M-1 sec-1) than with cardiac (M2) (1-5 x 10(6) M-1 sec-1) and NB-OK1 (M1) (1-5 x 10(6) M-1 sec-1) binding sites. At each receptor subtype, we observed no significant difference between the association rate constants of the R- and S-enantiomers of either QNB or QNB methiodide. Receptor stereoselectivity, when present, was associated with differences in unlabeled drug dissociation rate constants. The dissociation rate constant varied much more than the association rate constant, when either (R)-QNB dissociation from the three subtypes (half-life, 77 min to greater than 340 min; best fit, 40 days) or dissociation of the four drugs from each receptor subtype (half-lives varying from 1.4 min to 4 hr at M1 receptors, 1.1 to 77 min at M2 receptors, and 3.5 min to greater than 340 min at M3 receptors were obtained by competition kinetics analysis) was compared.
Asunto(s)
Miocardio/metabolismo , Páncreas/metabolismo , Parasimpatolíticos/metabolismo , Quinuclidinil Bencilato/análogos & derivados , Quinuclidinil Bencilato/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Unión Competitiva , Humanos , Técnicas In Vitro , Cinética , Neuroblastoma/metabolismo , Ratas , Estereoisomerismo , Tritio , Células Tumorales CultivadasRESUMEN
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.