Receptor最新文献

We have used the dissection of selected rat brain regions to compare the in vivo pharmacokinetics of [3H]QNB, (R,S)-[125I]-4IQNB, and (R,R)-[125I]-4IQNB binding to the muscarinic acetylcholine receptor (mAChR). [3H]IQNB is distributed in accordance with the m2 subtype concentration, (R,S)-[125I]-4IQNB is distributed in accordance with the total mAChR concentration, and (R,R)-[125I]-4IQNB is distributed in accordance with the m1/m4 subtype concentration. Although the cerebellum is relatively poor in mAChR (composed almost exclusively of the m2 subtype), the [3H]QNB concentration in the cerebellum is nearly equal to that in the other brain regions and is predominantly composed of specific binding. In contrast, the (R,S)-[125I]-4IQNB and (R,R)-[125I]-4IQNB concentrations in the cerebellum are relatively low and are predominantly or exclusively composed of nonspecific binding. These results dramatically demonstrate the in vivo m2 selectivity of [3H]QNB. All three radioligands exhibit large population standard deviations, with a substantial reduction of the between-animal variability resulting from normalization to each individual animal's corpus striatum value. Thus, the large population standard deviations arise from variability in radioligand delivery (variations in global cerebral blood flow, radioligand binding to serum proteins, loss of parent radioligand through conversion to metabolites, and blood-brain barrier transport.

Modulators are endogenous low-mol-wt inhibitors of glucocorticoid receptor activation, and are mimicked by exogenous sodium molybdate. Activation involves the dissociation of the 90-kDa heat-shock protein from the receptor. The heat-shock protein is thought to bind to a conserved 20-amino-acid region in the steroid-binding domain of the receptor (595-614 of the rat protein). Synthetic peptides corresponding to this amino acid sequence prevented the modulators and sodium molybdate from inhibiting receptor activation. These results imply that the region 595-614 of the rat glucocorticoid receptor is also a modulator/molybdate binding site.

Modulators-1 and -2 are endogenous low-mol-wt regulators of glucocorticoid and mineralocorticoid receptors and protein kinase C. Structural analysis of apparently purified modulators suggested that these molecules were novel ether aminophosphoglycerides. Subsequent X-ray crystallography and NMR spectroscopy indicated that the ultra-large scale modulator preparations were contaminated with glutamate and aspartate, although these amino acids lacked modulator activity. In this article, we describe the purification of modulator-2 from rabbit liver cytosol and the separation of this phosphoglyceride from these amino acids. This purification was similar to the ultra-large scale version (Bodine, P.V. and Litwack, G. [1990] J. Biol. Chem. 265, 9544-9554), but involved the chromatography of trypsinized rabbit liver cytosol on the 7-L bed volume Sephadex G-15 gel-filtration column. As before, two peaks of modulator activity (modulator-1 and -2), as well as a DNA-binding inhibitor (peak-3), eluted from the gel-filtration column. The resulting modulator-2 pool was incubated with glutamate decarboxylase and treated batch-wise with Dowex-50W cation-exchange resin and Chelex-100 resin. This enzyme/resin-treated modulator-2 preparation was then chromatographed on a Dowex-1 anion-exchange column. Finally, modulator-2 was purified by preparative silica TLC. This last purification step resulted in the separation of modulator-2 from glutamate, aspartate, and gamma-aminobutyrate. In summary, rabbit liver cytosol appears to be a reasonable source of modulator-2. In addition, treatment of the preparation with glutamate decarboxylase seems to facilitate the subsequent separation of modulator-2 from the contaminating amino acids.

ETs-induced contractions were resistant to ET(A)-selective antagonists and believed to be mediated by activation of ETB receptors in guinea pig bronchus. In the present study, the effects of the ET antagonists, PD143296 (Ac-D-Phe-L-Leu-L-Phe-L-Ile-L-Ile-L-Trp.2Na) and PD145065 (Ac-[(R)-2-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5-yl]Gly)-L-Leu-L-Asp-L-Ile-L-Ile- L-Trp.2Na), on contractions induced by ET-1, ET-3, sarafotoxin S6c (STXc), and IRL1620 in the isolated hilar bronchus of the guinea pig were investigated. An ETA/B nonselective antagonist, PD145065 antagonized contractions induced by ET-1, ET-3, STXc, and IRL1620. Its antagonistic activity against ET-1, with pKB of 5.77 +/- 0.02 (n = 16, 3-10 microM), was significantly lower than that against ET-3, with pKB of 6.18 +/- 0.02 (n = 12, 3-10 microM), STXc, with pKB of 5.97 +/- 0.01 (n = 14, 3-10 microM), and IRL1620, with pKB of 6.80 +/- 0.04 (n = 14, 0.3-1 microM). Conversely, although a putative ETB-selective antagonist, PD143296 (10 microM) slightly but significantly antagonized the concentration-response curve of IRL1620 (pKB = 5.28 +/- 0.14, n = 6), it had no effect on ET-1-,ET-3-, or STXc-induced contractions. These results suggest that ETs possibly activate ETB2 or an atypical ETB receptor subtype in guinea pig hilar bronchus.

Trypomastigotes of Trypanosoma cruzi were impermeable to exogenous radiolabeled ATP for up to 2 h at 4 degrees C. Radioligand binding assays in the cold showed that trypomastigotes had two populations of saturable ATP receptors (ATP-Rs) and the radioligand interaction was reversed by nonlabeled ATP in concentration-dependent assays. The Kds of high- and low-affinity ATP-Rs were 7.27 x 10(-8) and 4.32 x 10(6)M, respectively. The BmaxS for ATP-R1 and ATP-R2 were 2.05 x 10(-14) and 2.50 x 10(-12) mol/4 x 10(-6) flagellates, respectively, or 3100 ATP-R1 copies per trypomastigote and 376,000 ATP-R2 copies per trypomastigote. ATP,2-methyl-thio-ATP,ITP, and ADP displaced ATP-Rs (IC50s: 2.59 x 10(-6) to 7.84 x 10(-6)M/4 x 10(6) trypomastigotes). UTP, CTP, ADP beta S, Cibacron blue, and azido-ATP were 10-100 times less effective. Atractyloside, adenosine, suramin, cAMP, Basilen blue, and GTP failed to displace T. cruzi ATP-Rs. Infective vertebrate stage trypomastigote ATP-Rs were localized to a family of 63 kDa surface glycopolypeptides and ATP-Rs appeared to be stage-regulated because the noninfective insect epimastigote forms had ATP-Rs with KdS that are not capable of metabolic interactions with host-cell micromolar ATP levels. Trypomastigote ATP-Rs may play an important role in the induction of the process of T. cruzi intracellular parasitosis.

This work reviews evidence that some physiological and behavioral responses to steroid hormones use membrane-associated receptors. The review emphasizes research with an amphibian model, Taricha granulosa, but also cites examples from mammalian research. Many studies document steroid responses that occur within a time frame of a few milliseconds or minutes. In Taricha, corticosterone rapidly inhibits reproductive behaviors and causes site-specific changes in neurotransmitter concentrations and neuronal activity. Ligand binding assays using radiolabeled corticosterone and neuronal membranes from Taricha (and other animals) provide evidence that there are high-affinity steroid receptors in neuronal membranes. Subcellular fractionation, autoradiography, and immunocytochemistry add support to the conclusion that there are steroid receptors in neuronal membranes. Other studies indicate that, in Taricha and other animals, there are two types of membrane-associated steroid receptors--ligand-gated ion channels (specifically, the GABAA receptor) and G-protein coupled receptors.

R3T3 cells, a mouse fibroblast cell line, express the type 2 angiotensin II receptor (AT2), but not the AT1 subtype. We previously reported that expression of AT2 sites in these cells were regulated by various conditions: 1. The number of AT2 sites increased considerably when cells were contact-inhibited; 2. Stimulation of R3T3 cells with various mitogens caused a rapid decline of AT2 binding sites; and 3. Stimulation of cells with angiotensin ligands resulted in upregulation of the AT2 sites. In this study, to determine if altered AT2 expression is under transcriptional, posttranscriptional, or translational control, we examined the level of AT2 mRNA in R3T3 cells in response to various treatments. There was a 200-fold increase in AT2 mRNA levels in quiescent cells as compared to growing cells. Results from nuclear run-on assays suggested that the differences in AT2 mRNA levels were primarily caused by changes in the rate of AT2 gene transcription. Stimulation of cells with fibroblast growth factor caused an approximate threefold reduction of AT2 mRNA levels, and also increased the rate of degradation of AT2 mRNA, which correlated with the decrease in AT2 binding activity seen under these conditions. However, whereas treatment with angiotensin ligands increased AT2 binding activity, the level of AT2 transcripts did not increase. This pattern of expression implies that regulation of AT2 receptors occurs at multiple levels, involving translational and/or posttranslational as well as transcriptional control, and further affords the cell the ability to rapidly modulate the number of AT2 binding sites in response to changing extracellular conditions.

Recent pharmacological and functional studies have suggested the presence of more than one alpha-1 adrenergic receptor subtype in human corpus cavernosum (HCC). In this study, we sought to identify the alpha-1 adrenergic receptor (alpha 1-AR) subtypes expressed in HCC whole tissue and in trabecular smooth muscle subcultured from this tissue. We have utilized RNase protection assays and in situ hybridization (ISH) techniques to identify and localize these receptor subtypes. RNase protection assays of mRNA isolated from whole tissue demonstrated the presence of mRNA transcripts for three alpha 1-AR receptor subtypes (alpha 1d, alpha 1b, and alpha 1a). alpha 1d-AR and alpha 1a-AR appear to be more abundant than alpha 1b-AR. The identification and localization of mRNA for alpha 1-AR subtypes in whole tissue was demonstrated by RNA protection assays and ISH analysis. Immunocytochemical analysis of alpha 1-AR by an antipeptide antibody developed against a specific amino acid sequence derived from alpha 1d-AR subtype demonstrated specific staining of the smooth muscle cells, suggesting the expression of alpha 1d-AR subtype. In cultured HCC smooth muscle cells (HCC SMC), phenylephrine,alpha 1-AR agonist stimulated Na+/K+ ATPase activity, suggesting the presence of functional alpha 1-AR. RNase protection assay of mRNA isolated from HCC SMC grown in culture further demonstrated the presence of mRNA transcripts for alpha 1d-AR and alpha 1a-AR subtypes. ISH analysis and confocal microscopy also indicate that the SMC express the alpha 1d-AR and alpha 1a-AR subtypes. The data presented suggests that HCC and SMC derived from this tissue express at least three alpha 1-AR subtypes. Identification of these receptor subtypes should allow characterization of the functional role of these receptor subtypes in regulation of trabecular smooth muscle tone and penile detumescence.

To characterize the functional coupling of the beta 2-AR to the cardiac Ca2+ channel in a system with a single receptor subtype, we stably cotransfected a Chinese hamster fibroblast (CHW) cell line, which lacks beta 2-ARs and Ca2+ channels, with the rabbit cardiac Ca2+ channel alpha 1 and beta 2 subunits and the human beta 2-AR cDNAs. The effects of beta 2-AR stimulation on the expressed Ca2+ channel current were examined using the whole-cell patch-clamp technique. CHW cells transfected with the Ca2+ channel subunits displayed a voltage-dependent inward current having properties typical of native cardiac L-type Ca2+ channels. The expressed current was increased by a phosphorylation-dependent mechanism. CHW cells cotransfected with the Ca2+ channel subunits and the beta 2-AR were responsive to isoproterenol (Iso) in a dose-dependent manner. Iso (10 microM) increased peak Ca2+ channel current to 172 +/- 5% (n = 17) of control amplitude, indicating that the expressed Ca2+ channels are functionally coupled to the beta 2-AR. The results demonstrate unequivocally that beta 2-ARs can modulate the activity of cardiac Ca2+ channels, independent of beta 1-ARs. The results also demonstrate the usefulness of the CHW heterologous expression system, the first to reconstitute physiological modulation of an L-type Ca2+ channel by the beta 2-AR, for studying receptor subtype-specific regulation of the Ca2+ channel.

Insulin-like growth factor-I (IGF-I) is a pleiotropic hormone synthesized in a wide variety of cell types. As its name suggests, it is highly homologous to insulin and has many similar growth promoting effects. IGF-I is believed to be the major anabolic mediator of growth hormone action. A second insulin-like growth factor (IGF-II) is expressed at high levels in the fetus. IGF-II is homologous to IGF-I and interacts with the IGF-I (type 1) receptor as well as its own (type 2) receptor. Targeted manipulation of the mouse genome enables dissection of gene function in the context of the whole animal. We have generated strains of mice deficient in IGF-I production by homologous recombination that demonstrate important roles for IGF-I in both pre- and postnatal development.