RESUMEN
We disclose a new compound class of potent and selective alpha-1A adrenergic receptor antagonists exemplified by the geminally, disubstituted cyclic imide 7. The optimization of lead compounds resulting in the cyclic imide motif is highlighted. The results of in vitro and in vivo studies of selected compounds are presented.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1 , Animales , Perros , Semivida , Imidas/sangre , Imidas/síntesis química , Imidas/farmacocinética , Masculino , Piperidinas/síntesis química , Piperidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadAsunto(s)
Antagonistas Adrenérgicos alfa/síntesis química , Oxazoles/síntesis química , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Administración Oral , Antagonistas Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacocinética , Antagonistas Adrenérgicos alfa/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Unión Competitiva , Disponibilidad Biológica , Presión Sanguínea/efectos de los fármacos , Perros , Humanos , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Oxazoles/metabolismo , Oxazoles/farmacocinética , Oxazoles/farmacología , Presión , Próstata/efectos de los fármacos , Próstata/fisiología , Ensayo de Unión Radioligante , Ratas , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Uretra/efectos de los fármacos , Uretra/fisiologíaRESUMEN
A novel class of potent and selective alpha-1a receptor antagonists has been identified. The structures of these antagonists were derived from truncating the 4-aryl dihydropyridine subunit present in known alpha-1a antagonists. The design principles which led to the discovery of substituted phenylacetamides, the synthesis and SAR of key analogues, and the results of select in vitro and in vivo studies are described.
Asunto(s)
Acetamidas/farmacología , Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/farmacología , Acetamidas/química , Acetamidas/farmacocinética , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Perros , Relación Estructura-ActividadRESUMEN
A series of alpha1a receptor antagonists derived from a 4-aryl-3,4-dihydropyridine-2-one heterocycle is disclosed. Potency in the low nanomolar to picomolar range along with high selectivity was obtained. In vivo efficacy in a prostate contraction model in rats was observed with a few derivatives.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/farmacología , Dihidropiridinas/farmacología , Antagonistas Adrenérgicos alfa/química , Animales , Dihidropiridinas/química , RatasRESUMEN
alpha(1) Adrenergic receptors mediate both vascular and lower urinary tract tone, and alpha(1) receptor antagonists such as terazosin (1b) are used to treat both hypertension and benign prostatic hyperplasia (BPH). Recently, three different subtypes of this receptor have been identified, with the alpha(1A) receptor being most prevalent in lower urinary tract tissue. This paper explores 4-aryldihydropyrimidinones attached to an aminopropyl-4-arylpiperidine via a C-5 amide as selective alpha(1A) receptor subtype antagonists. In receptor binding assays, these types of compounds generally display K(i) values for the alpha(1a) receptor subtype <1 nM while being greater than 100-fold selective versus the alpha(1b) and alpha(1d) receptor subtypes. Many of these compounds were also evaluated in vivo and found to be more potent than terazosin in both a rat model of prostate tone and a dog model of intra-urethral pressure without significantly affecting blood pressure. While many of the compounds tested displayed poor pharmacokinetics, compound 48 was found to have adequate bioavailability (>20%) and half-life (>6 h) in both rats and dogs. Due to its selectivity for the alpha(1a) over the alpha(1b) and alpha(1d) receptors as well as its favorable pharmacokinetic profile, 48 has the potential to relieve the symptoms of BPH without eliciting effects on the cardiovascular system.
Asunto(s)
Antagonistas Adrenérgicos alfa/síntesis química , Pirimidinonas/síntesis química , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/farmacocinética , Antagonistas Adrenérgicos alfa/farmacología , Animales , Disponibilidad Biológica , Células CACO-2 , Cristalografía por Rayos X , Perros , Humanos , Masculino , Hiperplasia Prostática/tratamiento farmacológico , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 1/metabolismo , Relación Estructura-ActividadRESUMEN
Furo[3,4-d]pyrimidinones were found to be metabolites of dihydropyrimidinones such as 1a-b that are subtype-selective antagonists of the alpha1a-adrenergic receptor. A versatile synthesis that provides access to furo[3,4-d]pyrimidinones in high yield and in enantiomerically pure forms is described along with structure-activity relationships in the series.
Asunto(s)
Antagonistas Adrenérgicos alfa/síntesis química , Pirimidinonas/síntesis química , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Antagonistas Adrenérgicos alfa/farmacología , Animales , Unión Competitiva , Estructura Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Prazosina/metabolismo , Pirimidinonas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/síntesis química , Pirimidinonas/síntesis química , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Animales , Unión Competitiva , Presión Sanguínea/efectos de los fármacos , Perros , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Técnicas In Vitro , Masculino , Próstata/metabolismo , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 1/metabolismo , Estereoisomerismo , Uretra/efectos de los fármacos , Uretra/fisiologíaRESUMEN
Dihydropyrimidinones, such as 1, represent a novel class of alpha(1a) adrenoceptor antagonists with potential for the treatment of benign prostatic hyperplasia (BPH) (see part 1 of this series). Analysis of the metabolites of 1 revealed that 4-methoxycarbonyl-4-phenylpiperidine is formed as the major metabolite and is an agonist at the mu-opioid receptor. To circumvent any potential liability resulting from the metabolite, we decided to identify alternate templates devoid of agonist activity at the mu-opioid receptor to replace the 4-methoxycarbonyl-4-phenylpiperidine moiety. The present study describes the synthesis and SAR of dihydropyrimidinones linked to substituted 4-phenylpiperazine containing side chains. Compound (+)-38 was identified as a lead compound with a binding and functional profile comparable to that of 1. The putative metabolite 2-carboxamidophenylpiperazine has negligible affinity for the mu-opioid receptor.
Asunto(s)
Antagonistas Adrenérgicos alfa/síntesis química , Piperazinas/síntesis química , Pirimidinonas/síntesis química , Receptores Adrenérgicos alfa 1/metabolismo , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Animales , Unión Competitiva , Disponibilidad Biológica , Perros , Diseño de Fármacos , Proteínas de Unión al GTP/metabolismo , Semivida , Humanos , Técnicas In Vitro , Masculino , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacología , Próstata/metabolismo , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Ratas , Receptores Opioides mu/agonistas , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relación Estructura-ActividadAsunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/farmacología , Prazosina/análogos & derivados , Antagonistas Adrenérgicos alfa/síntesis química , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/metabolismo , Animales , Antihipertensivos/síntesis química , Antihipertensivos/química , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Humanos , Masculino , Especificidad de Órganos , Prazosina/síntesis química , Prazosina/química , Prazosina/metabolismo , Prazosina/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 1/metabolismo , Médula Espinal/metabolismo , Relación Estructura-ActividadRESUMEN
The anti-anxiety agent ipsapirone has been shown to have modest affinity for alpha-1 receptors. We disclose the discovery of potent alpha-1a receptor subtype selective antagonists based on the ipsapirone structure which possess selectivity versus the 5-HT receptors tested. These antagonists were obtained by tethering a saccharin ring to 4-phenyl-3-carboxyethyl piperidines. The design principles which led to this structural motif are discussed. The synthesis of key analogs, their SAR, as well as results of selected in vitro and in vivo studies are described.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/síntesis química , Pirimidinas/química , Animales , Ansiolíticos/química , Ansiolíticos/metabolismo , Diseño de Fármacos , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Humanos , Masculino , Modelos Químicos , Hiperplasia Prostática/tratamiento farmacológico , Pirimidinas/metabolismo , Ratas , Receptores Adrenérgicos alfa 1 , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Mice that are homozygous for the targeted disruption of the gene encoding the B2 bradykinin receptor have been generated. The gene disruption results in a deletion of the entire coding sequence for the B2 receptor. The disruption of the B2 receptor gene has been confirmed by genetic, biochemical, and pharmacological analyses. Mice that are homozygous for the disruption of the B2 receptor gene are fertile and indistinguishable from their littermates by visual inspection. Bradykinin fails to produce responses in pharmacological preparations from ileum, uterus, and the superior cervical ganglia from these mice. Therefore, expression of a single gene appears to be responsible for conferring responsiveness to bradykinin in these tissues.
Asunto(s)
Bradiquinina/farmacología , Músculo Liso/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de Bradiquinina/genética , Animales , Secuencia de Bases , Femenino , Marcación de Gen , Ratones , Datos de Secuencia Molecular , Receptores de Bradiquinina/fisiologíaRESUMEN
A preliminary characterization is provided of a naturally occurring cyclic peptide with interesting and potent biological activity. A 31-residue cyclic peptide, designated cyclopsychotride A [1], was obtained from the organic extract of the tropical plant, Psychotria longipes. Compound 1 inhibited [125I] neurotensin (NT) binding to HT-29 cell membranes (IC50 3 microM) and also stimulated increased levels of cytosolic Ca2+ in two unrelated cell lines that do not express NT receptors. The peptide was found to dose-dependently increase intracellular Ca2+ at concentrations ranging from 3 to 30 microM, and this response was not blocked by a known NT antagonist. Cyclopsychotride A [1] possesses three disulfide linkages and is thought to be the largest cyclic peptide isolated from a natural source. Both 1H-nmr and cd spectroscopy showed 1 to be highly structured.
Asunto(s)
Ciclotidas , Neurotensina/antagonistas & inhibidores , Péptidos Cíclicos/aislamiento & purificación , Plantas Medicinales/química , Secuencia de Aminoácidos , Brasil , Calcio/metabolismo , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos Cíclicos/farmacología , Conformación Proteica , Espectrometría de Masa Bombardeada por Átomos Veloces , Células Tumorales CultivadasRESUMEN
A cDNA clone encoding a human B1 bradykinin receptor was isolated from a human embryonic lung fibroblast cDNA library by expression cloning. The photoprotein aequorin was utilized as an indicator of the ability of the B1 receptor agonist [des-Arg10]kallidin to mediate Ca2+ mobilization in Xenopus laevis oocytes injected with RNA. A clone was isolated with a 1307-nucleotide insert which contains an open reading frame encoding a 353-amino acid protein with the characteristics of a G-protein-coupled receptor. The amino acid sequence of the B1 bradykinin receptor is 36% identical to the amino acid sequence of the B2 bradykinin receptor. The cloned B1 bradykinin receptor expressed in mammalian cells exhibits high affinity binding for 3H-labeled [des-Arg10]kallidin and low affinity for bradykinin. The B1 receptor antagonist [des-Arg10,Leu9]kallidin effectively displaces 3H-labeled [des-Arg10]kallidin from the cloned receptor, whereas the B2 receptor antagonist Hoe-140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, where Thi is L-[3-(2-thienyl)alanyl], Tic is D-(1,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl), and Oic is L-[(3aS, 7aS)-octahydroindol-2-yl-carbonyl]) does not. Therefore, the expressed receptor has the pharmacological characteristics of the B1 receptor subtype. The availability of both the cloned human B1 and B2 bradykinin receptors should allow the elucidation of the relative contributions of these two receptor subtypes in acute and chronic inflammatory processes.
Asunto(s)
Receptores de Bradiquinina/genética , Receptores de Bradiquinina/metabolismo , Secuencia de Aminoácidos , Animales , Bradiquinina/análogos & derivados , Bradiquinina/metabolismo , Clonación Molecular , ADN Complementario/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1/farmacología , Calidina/análogos & derivados , Calidina/metabolismo , Datos de Secuencia Molecular , Oocitos , Receptores de Bradiquinina/clasificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
A human B2 bradykinin receptor cDNA was cloned from the lung fibroblast cell line, CCD-Lu. This clone was utilized to isolate a genomic clone of a mouse B2 bradykinin receptor. Both clones encode a protein that has the predicted characteristics of a seven transmembrane domain G-protein-coupled receptor. The DNA sequence of these two clones is 84% identical in the putative coding region. The clones have been heterologously expressed in a mammalian cell line lacking endogenous bradykinin receptors, COS-7, and a comparative analysis of their pharmacology was done. Both clones exhibit properties characteristic of the B2 bradykinin receptor, binding bradykinin with high affinity (KD = 0.1-0.2 nM) and binding des-Arg9 bradykinin with a very low affinity (IC50 > 5 microM). Interestingly, the mouse B2 bradykinin receptor has a 60-80 fold higher affinity than the human B2 bradykinin receptor for the peptide antagonists D-Arg0[Hyp3,Thi5,8,D-Phe7]bradykinin and D-Arg0[Hyp3,D-Phe7]bradykinin.
Asunto(s)
Receptores de Bradiquinina/genética , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Ratas , Receptores de Bradiquinina/metabolismo , Especificidad de la EspecieRESUMEN
Kinin B1 receptors on rabbit aorta smooth muscle cells in culture were investigated. [3H]Des-Arg10-kallidin labeled a single site in cells at early passage with an equilibrium dissociation constant of 258 pM and a maximal binding density of approximately 680 sites/cell. Treatment of the same cells for 18 h with epidermal growth factor increased the binding density over 6-fold without affecting the ligand's affinity. At latter passages, the density of binding sites was found to increase and the growth factor had a much less pronounced effect. The rank order of potencies for agonist inhibition of binding (des-Arg10-kallidin > des-Arg9-BK = kallidin > bradykinin) was consistent with the specific labeling of a B1 receptor. Also, [3H]des-Arg10-kallidin binding was potently inhibited by the B1 receptor antagonist des-Arg9[Leu8]bradykinin but not by the B2 receptor antagonist Hoe 140. The agonists were found to stimulate phosphoinositide hydrolysis in the smooth muscle cells with an order of potencies that reflected their binding assay activities. Des-Arg9[Leu8] BK blocked the des-Arg10-kallidin response with a potency consistent with its known B1 receptor activity while Hoe 140 was inactive. These results demonstrate the presence of inducible B1 receptors on rabbit aorta smooth muscle cells in culture that couple to phospholipase C activation. These cells should be useful in future studies of the mechanisms and factors involved in the regulation of expression of the B1 receptor.
Asunto(s)
Bradiquinina/análogos & derivados , Calidina/análogos & derivados , Músculo Liso Vascular/metabolismo , Receptores de Bradiquinina/metabolismo , Animales , Aorta/metabolismo , Sitios de Unión , Unión Competitiva , Bradiquinina/metabolismo , Bradiquinina/farmacología , Células Cultivadas , Activación Enzimática/fisiología , Hidrólisis , Calidina/metabolismo , Calidina/farmacología , Lisofosfolipasa/metabolismo , Músculo Liso Vascular/citología , Fosfatidilinositoles/metabolismo , Conejos , Receptores de Bradiquinina/efectos de los fármacosRESUMEN
The pharmacology of cloned B2 bradykinin receptors heterologously expressed in cell lines lacking any endogenous bradykinin receptors was analyzed. The possibility of B2 bradykinin receptor heterogeneity had been proposed on the basis of numerous studies in various tissue preparations. The results reported here permit a direct evaluation of some of these hypotheses by examining the pharmacological properties of cloned bradykinin receptors. A cloned human B2 bradykinin receptor was stably transfected into Chinese hamster ovary cells. The data suggest that in response to bradykinin (BK), the cloned receptor activates both phosphatidylinositol hydrolysis and arachidonic acid release by independent pathways. Thus, the activation of these two second messenger pathways does not require the existence of two B2 bradykinin receptor subtypes. A mouse gene encoding the B2 bradykinin receptor was isolated, and the coding region was expressed in COS-7 cells. This murine receptor exhibited the pharmacological properties of a "classical" B2 bradykinin receptor. A comparison of the pharmacological profiles of cloned human and murine homologs of the B2 bradykinin receptor indicates that both receptors bind agonists with similar properties. However, the two receptors differ dramatically in their affinity for some peptide antagonists. The mouse receptor has a 60- to 80-fold higher affinity for [D-Arg0Hyp3, Thi5,8,D-Phe7]BK and [D-Arg0,Hyp3,D-Phe7]BK than its human homolog. Thus, the species of a bradykinin receptor can have a significant effect on its pharmacology. The cloning, expression, and pharmacological comparison of human and mouse B2 bradykinin receptor genes indicate that some of the previous reports of B2 receptor subtypes can be explained by species differences in a single B2 bradykinin receptor gene.
Asunto(s)
Receptores de Bradiquinina/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Receptores de Bradiquinina/genética , Receptores de Bradiquinina/metabolismo , Especificidad de la EspecieRESUMEN
A high affinity radioligand for bradykinin B2 receptors was prepared by coupling an activated ester of [125I]4-iodobenzoic acid to the amino terminus nitrogen of the potent B2 antagonist HOE 140. The ligand, [125I]para-iodophenyl HOE 140 ([125I]PIP HOE 140), bound to a homogeneous set of sites in guinea pig ileal membranes with an equilibrium dissociation constant of 15 pM and a maximal binding density of 193 fmole/mg protein. Competition studies with a number of BK-related peptides indicated that the ligand specifically labeled B2 receptors in the preparation. The results suggest that [125I]PIP HOE 140 will be a useful tool for future studies of B2 receptors.
Asunto(s)
Bradiquinina/análogos & derivados , Íleon/metabolismo , Receptores de Bradiquinina/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Bradiquinina/antagonistas & inhibidores , Bradiquinina/síntesis química , Bradiquinina/metabolismo , Bradiquinina/farmacología , Membrana Celular/metabolismo , Cobayas , Radioisótopos de Yodo , Cinética , Masculino , Datos de Secuencia Molecular , Músculo Liso/metabolismo , Ensayo de Unión Radioligante , Receptores de Bradiquinina/efectos de los fármacos , Receptores de Bradiquinina/aislamiento & purificaciónRESUMEN
Bradykinin (BK) B2 receptors in guinea pig ileum were characterized in both membrane and soluble form. [3H]BK bound to a single class of sites with almost identical affinities in membranes prepared from the longitudinal muscle, circular muscle and mucosal layers of the ileum. The pharmacology of the binding in the distinct layers was indistinguishable. The detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) maximally solubilized nearly 80% of membrane binding activity in a very stable conformation. In soluble preparations, [3H]BK labeled a single class of sites but with about 10-fold lower affinity. The affinities of BK analogs in competition studies were similarly reduced. There was no difference in the pharmacology of the binding in soluble receptors prepared from the different layers of the ileum. The results show that the ileum is a good source of solubilized B2 receptors and that the receptors in the smooth muscle and the mucosa are very similar.
Asunto(s)
Bradiquinina/análogos & derivados , Mucosa Intestinal/metabolismo , Músculo Liso/metabolismo , Receptores de Neurotransmisores/metabolismo , Secuencia de Aminoácidos , Animales , Bradiquinina/metabolismo , Membrana Celular/metabolismo , Cobayas , Íleon , Masculino , Datos de Secuencia Molecular , Receptores de Bradiquinina , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/aislamiento & purificación , SolubilidadRESUMEN
A human BK-2 bradykinin receptor was cloned from the lung fibroblast cell line CCD-16Lu. The cDNA clone encodes a 364 amino acid protein that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The predicted amino acid sequence of the human BK-2 receptor is 81% identical to the smooth muscle rat BK-2 receptor (1). Transfection of the human BK-2 receptor cDNA into COS-7 cells results in the expression of high levels of specific BK binding sites. Saturation binding analysis indicates that the human BK-2 receptor expressed in COS-7 cells binds BK with a KD of 0.13 nM. Pharmacological characterization of the expressed BK receptor is consistent with the cDNA encoding a receptor of the BK-2 subtype. The BK-2 receptor antagonist Hoe 140 (2), D-Arg0[Hyp3, Thi5, D-Tic7, Oic8]BK has a high affinity (IC50 = 65 pM) for the cloned human receptor. The tissue distribution of the human BK-2 receptor was analyzed by competitive PCR with human tissue cDNA and is similar to that determined for the BK-2 receptor in the rat.
Asunto(s)
Bradiquinina/metabolismo , Receptores de Neurotransmisores/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Clonación Molecular , Glicosilación , Humanos , Cinética , Pulmón , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Bradiquinina , Receptores de Neurotransmisores/metabolismo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
1. Bradykinin (BK)-induced contraction of ileal smooth muscle is assumed to be due to phosphoinositide hydrolysis but this has never been reported. We have investigated whether BK receptors are linked to this transduction mechanism in guinea-pig ileum longitudinal muscle and determined whether these receptors are equivalent to those labelled in [3H]-BK binding assays. 2. In membranes prepared from longitudinal muscle, [3H]-BK bound to a single class of sites with high affinity. Characterization of the binding with BK analogues indicated that the radioligand selectivity labelled a B2 type receptor. 3. BK significantly elevated tissue levels of [3H]-inositol phosphates in longitudinal muscle slices preincubated with [3H]-myo-inositol. The agonists potencies of BK, Lys-BK, Met-Lys-BK, Tyr5-BK and Tyr8-BK were in agreement with their relative potencies in the binding assay. The B1 receptor agonist des-Arg9-BK, did not stimulate inositol phosphate production. The response to BK was blocked by known B2 receptor antagonists but not by the B1 antagonist des-Arg9, Leu8-BK. 4. BK-induced phosphoinositide hydrolysis was unaffected by exposure of muscle slices to either atropine or indomethacin. 5. The results indicate that the B2 receptors linked to phosphoinositide turnover in ileal longitudinal muscle exhibit properties similar to those involved in contractile responses. Also, the receptor mediating the phosphoinositide response is likely to be that labelled in the [3H]-BK binding studies.