RESUMEN
Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.
Asunto(s)
Calcio/metabolismo , Receptores de Neuroquinina-3/fisiología , Secuencia de Aminoácidos/fisiología , Animales , Clonación Molecular/métodos , Humanos , Ratones , Datos de Secuencia Molecular , Neuroquinina A/metabolismo , Neuroquinina A/farmacología , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacología , Receptores de Neuroquinina-3/efectos de los fármacosRESUMEN
Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.
Asunto(s)
Neuropéptidos/metabolismo , Oligopéptidos/metabolismo , Receptores de Neuropéptido/metabolismo , Secuencia de Aminoácidos , Arrestinas/metabolismo , Secuencia de Bases , Calcio/metabolismo , Línea Celular , FMRFamida/farmacología , Humanos , Ligandos , Datos de Secuencia Molecular , Receptores de Neuropéptido/genética , beta-ArrestinasRESUMEN
A cDNA was cloned from a rabbit spleen cDNA library which encoded a G-protein alpha subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Galpha16 and mouse G(alpha)15, respectively. The rabbit G(alpha)subunit cDNA was subcloned into a mammalian expression vector and transiently co-transfected into HEK-293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G alpha subunit behaved similarly to G(alpha)15 or G(alpha)16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit G(alpha) subunit appears to be the ortholog of human G(alpha)16 and mouse G(alpha)15.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de la Membrana , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Clonación Molecular , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Biblioteca de Genes , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Ratones , Datos de Secuencia Molecular , Conejos , Receptor de Anafilatoxina C5a , Receptores de Complemento/genética , Receptores Opioides/genética , Alineación de Secuencia , Bazo/metabolismo , Transfección , Receptor de NociceptinaRESUMEN
The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.
Asunto(s)
Proteínas de la Membrana , Receptores de Leucotrienos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Clonación Molecular , Humanos , Leucotrieno D4/farmacología , Datos de Secuencia Molecular , Toxina del Pertussis , Receptores de Leucotrienos/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed W138 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and W138 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and W138 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed W138 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylated RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.
Asunto(s)
Línea Celular Transformada/metabolismo , Endotelina-1/metabolismo , Fibroblastos/metabolismo , ARN Mensajero/metabolismo , Receptores de Endotelina/metabolismo , Células 3T3 , Animales , Unión Competitiva , Northern Blotting , Calcio/metabolismo , Regulación hacia Abajo , Genes ras/genética , Humanos , Ratones , Virus 40 de los Simios/genéticaRESUMEN
This study demonstrates the presence of a novel endothelin (ET) receptor subtype that displays high affinity for both ETA- and ETB-selective ligands. This subtype has been identified in canine spleen membranes using ETB-selective agonists ET-3, IRL-1620, sarafotoxin 6c (S6c) as well as ETA-selective antagonists BQ123 and related cyclic pentapeptides. Binding of 125I-ET-3 to canine spleen membranes was specific and saturable with an apparent dissociation constant of 130 pM and maximum binding (Bmax) of 240.0 fmol/mg protein. Although the apparent affinities obtained with 125I-ET-1 and 125I-ET-3 were comparable (90 and 130 pM, respectively), the maximum binding obtained with 125I-ET-3 was approximately 35% of that obtained with 125I-ET-1, which indicates that canine spleen possesses both ETA and ETB receptors in the ratio 65:35. Competition binding experiments using 125I-ET-3 and unlabeled ET-1, ET-3, S6c, and IRL-1620 suggested that although ET-1 and ET-3 displayed similar high affinity, S6c and IRL-1620 were 20-300-fold weaker than ET-1 and ET-3 in competing for 125I-ET-3 binding to canine spleen membranes. In addition, BQ123, an ETA-selective antagonist, displaced 125I-ET-3 binding from canine spleen with an IC50 value of 30 nM. Similar profiles were obtained with related cyclic pentapeptides. Electrophysiological studies performed on Xenopus laevis oocytes injected with canine spleen poly(A)+ RNA indicated that the ETB receptor present in these tissues is functional and displays the same pharmacology as that observed in binding studies using these membranes. As a comparison, both binding and functional studies were performed in canine lung and the data indicate that the ETB receptor present in this tissue is similar to that of the cloned human ETB receptor but different from that present in canine spleen. These observations were further confirmed by performing cross-linking experiments on these membranes. Although canine lung and cloned human ETB receptors displayed the same molecular weight bands with similar pharmacology, canine spleen ETB receptors displayed different molecular weight bands and different pharmacology. In addition, the ETB receptors present in canine spleen were also identified in canine bladder, monkey spleen and human spleen. Thus, the data presented in this manuscript provide evidence for the presence of a novel ETB receptor in different tissues as well as different species including human.
Asunto(s)
Endotelinas/metabolismo , Receptores de Endotelina/metabolismo , Animales , Células CHO , Cricetinae , Perros , Femenino , Haplorrinos , Humanos , Radioisótopos de Yodo , Pulmón/metabolismo , Unión Proteica , Ensayo de Unión Radioligante , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/agonistas , Receptores de Endotelina/clasificación , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Bazo/metabolismo , Xenopus laevisRESUMEN
We have identified a novel integrin beta3 subunit, termed beta3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3'-untranslated region of the beta3C subunit differs from the previously reported beta3A (platelet) and beta3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The beta3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the beta3A gene. HEK 293 cells were stably co-transfected with alphaV and either beta3C (HEKbeta3C) or beta3A (HEKbeta3A). The viability of HEKbeta3C cells was lower than that of HEKbeta3A cells, and HEKbeta3C cells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both alphaVbeta3A and alphaVbeta3C isoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKbeta3A, HEKbeta3C cells failed to adhere to osteopontin, an alphaVbeta3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the beta3 integrin in cell adhesion and suggest a potential role for the beta3C integrin subunit in modulating cell-matrix interactions.
Asunto(s)
Antígenos CD/genética , Glicoproteínas de Membrana Plaquetaria/genética , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/fisiología , Secuencia de Bases , Northern Blotting , Adhesión Celular , Clonación Molecular , Humanos , Inmunohistoquímica , Integrina beta3 , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/fisiología , Ratas , TransfecciónRESUMEN
The Xenopus laevis oocyte has been widely utilized for cloning and functional expression of G-protein coupled receptors (GPCR). This system was used for the functional expression and characterization of the recently identified human C3a receptor. Complementary RNA from the human C3a receptor was transcribed in vitro and microinjected into Xenopus oocytes for functional characterization. A positive response to a synthetic C3a peptide agonist and to C3a, but not to platelet activating factor or fMetLeuPhe was detected. In addition, a response of approximately one third the amplitude obtained with C3a was obtained with rC5a. Conversely, oocytes co-injected with the C5a receptor and total RNA isolated from U937 cells responded to C5a as well as to C3a and the C3a synthetic peptide. A functional response with the anaphylatoxin C3a receptor in oocytes was dependent on co-injection of a pertussis toxin sensitive complementary human factor which could be supplied by co-injection of total RNA isolated from U937 cells. Oocytes expressing the anaphylatoxin C3a and C5a receptors responded to both agonists, in each case the response to the cognate ligand was substantially more robust than the response elicited by the other anaphylatoxin.
Asunto(s)
Antígenos CD/fisiología , Proteínas de la Membrana , Oocitos/fisiología , Receptores de Complemento/fisiología , Secuencia de Aminoácidos , Anafilaxia , Animales , Antígenos CD/biosíntesis , Antígenos CD/efectos de los fármacos , Complemento C3a/farmacología , Complemento C5a/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacología , Oocitos/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Toxina del Pertussis , Receptor de Anafilatoxina C5a , Receptores de Complemento/biosíntesis , Receptores de Complemento/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transcripción Genética , Factores de Virulencia de Bordetella/farmacología , Xenopus laevisRESUMEN
In a human neutrophil cDNA library, an orphan G-protein-coupled receptor, HNFAG09, with 37% nucleotide identity to the C5a receptor (C5a-R, CD88) was identified. A novel feature of this gene, unlike C5a-R and other G-protein-coupled receptors, is the presence of an extraordinarily large predicted extracellular loop comprised of in excess of 160 amino acid residues between transmembrane domains 4 and 5. Northern blot analysis revealed that expression of mRNA for this receptor in human tissues, while similar, was distinct from C5a-R expression. Although there were differences in expression, transcripts for both receptors were detected in tissues throughout the body and the central nervous system. Mammalian cells stably expressing HNFAG09 specifically bound 125I-C3a and responded to a C3a carboxyl-terminal analogue synthetic peptide and to human C3a but not to rC5a with a robust calcium mobilization response. HNFAG09 encodes the human anaphylatoxin C3a receptor.
Asunto(s)
Complemento C3a/metabolismo , Proteínas de la Membrana , Receptores de Complemento/genética , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Expresión Génica , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Péptidos/química , ARN Mensajero/genética , Ratas , Receptor de Anafilatoxina C5a , Proteínas Recombinantes , TransfecciónRESUMEN
Endothelin (ET) receptors display subtype heterogeneity and so far three subtypes of ET receptors, namely ETA, ETB, and ETC, have been identified, cloned, sequenced, and characterized. Based on the binding profile of ET and related peptides, a novel ET receptor (ETAX) was identified in the follicular membranes of Xenopus laevis oocytes (Kumar, C. S., Nuthulaganti, P., Pullen, M., and Nambi, P. (1993). Mol. Pharmacol. 44, 153-157). Here we report the cloning and characterization of this ETAX subtype from X. laevis heart. A cDNA was isolated that encodes a protein of 415 amino acids that shares 74, 60, and 51% identities with human ETA, human ETB, and Xenopus ETC receptors, respectively. Competition binding studies of the cloned receptor expressed in COS cells using ET-related peptides suggested that this receptor is pharmacologically identical to that expressed in Xenopus oocyte follicular, heart, and lung membranes. Phosphoinositide turnover and oocyte electrophysiological studies indicated that the cloned receptor is functionally coupled to a second messenger system.
Asunto(s)
Miocardio/metabolismo , Receptores de Endotelina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Southern Blotting , Células Cultivadas , Clonación Molecular , ADN Complementario , Humanos , Ligandos , Potenciales de la Membrana , Datos de Secuencia Molecular , Oocitos/fisiología , Receptores de Endotelina/metabolismo , Xenopus laevisRESUMEN
High-affinity (104 +/- 18 pmol/l) and high-density (204 +/- 25 fmol/mg) angiotensin II (AII) binding sites have been identified in Xenopus laevis heart membranes. Competition binding of [125I]Sar1,Ile8 angiotensin (SIA) to these receptors by peptide analogs selective for the mammalian AII receptor subtypes AT1 and AT2 suggested that the amphibian AII binding sites were more closely related to the AT1 receptor subtype. Also in common with AT1 receptors, dithiothreitol and GTP gamma S inhibited [125I]SIA binding to Xenopus heart receptors, exhibiting IC50 values of 600 and 0.95 mumol/l, respectively. In addition, Xenopus oocytes injected with Xenopus heart mRNA were capable of mobilizing calcium when exposed to AII, demonstrating that Xenopus AII receptors are functionally linked to a second-messenger system similar to that coupled to mammalian AT1 receptors. However, in contrast to both AT1 and AT2 receptor subtypes, nonpeptide antagonists DUP 753 and SK&F 108566 (AT1 receptor selective) and PD123319 (AT2 selective) did not bind the Xenopus AII receptors, thus establishing that the amphibian receptors were pharmacologically unique. Together, these results demonstrate that Xenopus heart AII receptors are functionally similar to mammalian AT1 receptors but are pharmacologically distinct from both AT1 and AT2 receptors.
Asunto(s)
Miocardio/metabolismo , Receptores de Angiotensina/metabolismo , Xenopus laevis/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Animales , Unión Competitiva , Calcio/metabolismo , Ditiotreitol/farmacología , Femenino , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
A Xenopus laevis heart cDNA library was screened using the human angiotensin type 1 (AT1) receptor cDNA coding sequence as a hybridization probe. A cDNA was isolated that encodes a protein of 363 amino acids that shares 63% sequence identity with the human AT1 receptor. Radioligand binding studies with the cloned receptor expressed in COS cells indicated that it is an angiotensin II receptor that possesses pharmacological properties distinct from those of the two known mammalian receptor subtypes, AT1 and AT2. Electrophysiological studies with the recombinant receptor expressed in X. laevis oocytes revealed that the amphibian receptor, like the mammalian AT1 receptor, can functionally couple to a second messenger system, leading to the mobilization of intracellular stores of calcium. However, nonpeptide antagonists selective for the mammalian AT1 and AT2 receptors do not block angiotensin II-stimulated functional responses in injected oocytes, which confirms that the amphibian receptor is a pharmacologically unique angiotensin II receptor. Nevertheless, based on conservation of structural features and motifs and similarity in coupling mechanisms, we speculate that the cloned Xenopus receptor is the amphibian counterpart of the mammalian AT1 receptor, having acquired its unique pharmacology as a consequence of evolutionary divergence.
Asunto(s)
Miocardio/metabolismo , Receptores de Angiotensina/metabolismo , Secuencia de Aminoácidos , Angiotensinas/metabolismo , Angiotensinas/farmacología , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN/química , ADN/genética , Electrofisiología , Modelos Moleculares , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Estructura Secundaria de Proteína , ARN Mensajero/química , ARN Mensajero/genética , Ensayo de Unión Radioligante , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Receptores de Angiotensina/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Transfección , Xenopus laevisRESUMEN
Follicular oocytes from Xenopus laevis display saturable and high affinity endothelin (ET)-1 binding sites. Competition binding experiments using unlabeled ET-1, ET-3, and sarafotoxin 6c indicated that these ET receptors belong to the ETA receptor subtype. However, the ETA receptor-selective antagonist BQ123 [cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu)] failed to inhibit 125I-ET-1 binding to these receptors, suggesting that these receptors belong to a novel subtype of ETA receptors (ETAX) distinct from the human ETA receptors. These endogenous receptors are present in the follicular layer, because defolliculated oocytes did not show any 125I-ET-1 binding. Addition of ET-1 to follicular oocytes led to Ca2+ mobilization, which was reversibly blocked by treatments that uncouple gap junctions, suggesting that these ETAX receptors mediate their response by transferring signals through gap junctions. On the other hand, the expressed human ETA receptor-mediated Ca2+ mobilization was not blocked by inhibitors of gap junctions. In agreement with the binding data, the endogenous ETAX receptor-mediated response was not inhibited by BQ123 even at 100 nM, whereas the expressed human ETA receptor-mediated response was inhibited by 50% at concentrations as low as 10 nM. This further confirms that the amphibian ETAX receptors are different from mammalian ETA receptors. Finally, ET-1 enhanced the rate of progesterone-induced maturation of follicular oocytes, implying the involvement of these endogenous ETAX receptors in an in vivo maturation process.
Asunto(s)
Calcio/fisiología , Oocitos/metabolismo , Receptores de Endotelina/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Electrofisiología , Endotelinas/metabolismo , Femenino , Uniones Intercelulares/fisiología , Datos de Secuencia Molecular , Ensayo de Unión Radioligante , Receptores de Endotelina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcripción Genética , Xenopus laevisRESUMEN
Steroid hormones have been shown to modulate a number of physiological processes in addition to their potent antiinflammatory effects. Endothelin (ET) is a newly discovered vasoconstrictor that is synthesized and released by endothelial cells and acts on adjacent vascular smooth muscle cells by interacting with specific cell surface receptors. Proinflammatory agents such as thrombin and transforming growth factor beta have been shown to up-regulate ET gene expression in vascular endothelial cells. We wondered whether the anti-inflammatory steroids might have any regulatory effect on the ET receptors present in the vascular smooth muscle cells. Rat vascular smooth muscle cells (A-10 cell line, ATCC.CRL 1476) were used as a model system to study the effects of glucocorticoids on ET receptor expression and function. These cells display high density and high affinity ET receptors that belong to the ETA subtype. Pretreatment of these cells with dexamethasone reduced the number of ET receptors by 50-60% without changing the affinity. Of the steroids tested, dexamethasone was most effective followed by prednisolone and hydrocortisone. Aldosterone, a mineralocorticoid, was 5000-fold less potent than dexamethasone. This effect of dexamethasone was dependent on the time of pretreatment and concentration of the steroid used. This down-regulation of ET receptors was also accompanied by an attenuated response to ET-1 in dexamethasone-pretreated cells. The inhibitory effect of dexamethasone was selective for ET receptors because the vasopressin-mediated response was unaffected. In addition, dexamethasone pretreatment of these cells resulted in 50-60% reduction in the steady-state level of ETA receptor mRNA as revealed by Northern analysis. These results suggest that glucocorticoid pretreatment of smooth muscle cells resulted in the down-regulation of the ETA receptor at the mRNA level.
Asunto(s)
Dexametasona/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Endotelinas/metabolismo , Expresión Génica , Técnicas In Vitro , ARN Mensajero/genética , Ratas , Receptores de Superficie Celular/genética , Receptores de Endotelina , Factores de TiempoRESUMEN
A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.