Journal of receptor research最新文献

New radioimagers, the HRRI (high resolution radioimager) and the Phosphorimager (phosphor screen : PS), apt to display more ample linear dose-response scale than radio-sensitive films, were tested in comparison with quantitative autoradiography (QA). GnRH receptor saturation experiments were achieved on tissue sections (rat pituitary, rat brain, human ovary) with a iodinate GnRH agonist (125I-[D-Ala6,Des-Gly10]-LH-RH Ethylamide) for determination of affinity constant (Kd). In rat pituitary, comparable results were obtained with the 3 methods (Kd: 0.4 to 0.6 nM). Discrepancies occurred in the hippocampus and in the granulosa cell layer of the preovulatory follicle, due to low resolutive (PS) or short linear dose-response (films) performances. In the hippocampus GnRH receptor affinity was under-estimated with PS (Kd: 2.3 vs 0.5 and 0.6 nM for QA and HRRI respectively). In the follicular granulosa cell layer it was over-estimated by QA (0.5 vs 50 nM for the HRRI), while PS did not allow resolution of this thin cell layer. In conclusion, the HRRI is a very powerful tool for the quantification of in situ radioligand binding (binding sites study and in situ hybridization) in very discrete areas.

The binding characteristics of [3H]quinuclidinyl benzilate ([3H]QNB) to isolated crude membranes of cultured bovine aortic endothelial cells were investigated. [3H]QNB bound to endothelial cell membranes with high affinity (kD = 0.056 nM) and limited capacity (132 fmol/mg DNA). The binding specificity, order of affinity and inhibition constants (Ki) were determined by displacement of bound [3H]QNB with unlabeled ligands. The order of affinity was QNB > atropine > 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) > p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) (M3 antagonist) > pirenzepine (M1 antagonist) > AFDX-116 (M2 antagonist) > (4-hydroxy-2-butynyl) trimethylammonium chloride m-chlorocarbanilate (McN-A-343, M1 agonist). These observations suggest that muscarinic receptors of endothelial cells in culture are likely to be of M3 and M1 subtype. Northern blot analysis of receptor subtypes using cDNA probes did not provide conclusive results due to the low level expression of these receptors in cultured cells. Solubilization of protein bound [3H]QNB with 1% digitonin and 0.02% cholate followed by analysis on sucrose density gradients demonstrated the presence of a specifically bound [3H]QNB-protein complex sedimenting at the 6.2S region of the gradient. These data demonstrate the presence of muscarinic acetylcholine receptor protein in cultured bovine aortic endothelial cells.

The structure of bacteriorhodopsin was used as a template to generate a model for G-protein coupled receptors. However, these receptors and the template are not related by sequence homology. Therefore a pragmatic and reproducible approach was developed to achieve an energetically favourable accommodation of receptor sequences to the backbone structure of bacteriorhodopsin. Improved interaction energy differences are used in a two step procedure analogous to a hypothetical folding mechanism for integral membrane proteins. The resulting model is in good agreement with existing data from structure-function studies.

alpha 2- and beta-adrenergic receptors in human placental membranes have been investigated using the radioligands [3H]-RX 821002 and [3H]-dihydroalprenolol, respectively. The specific binding of the alpha 2-adrenoceptor antagonist RX 821002 confirms the presence of an alpha 2-adrenoceptor in the human placenta, which has been characterized previously with [3H]-rauwolscine. The major finding presented here is a correlation between the alpha 2- and beta-adrenergic receptor concentrations (r = 0.765) in the human placenta at term. It is suggested that the alpha 2/beta adrenoceptor balance may play an important role in regulation of the vascular bed of the placenta. Determination of the alpha 2/beta ratio may help towards an understanding of the contractility of the placental vascular muscles.

Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein. In digitonin-permeabilized MA-10 Leydig tumor cells incubated with [gamma-32P]ATP, phosphorylation of MBR was detectable during treatment of the cells with dibutyryl cAMP. In conclusion, these data show that the MBR protein is an in vitro and in situ substrate of Protein kinase A, but the role of this phosphorylation in the regulation of steroidogenesis remains to be established.

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[3H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the beta-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.

We describe a simple nonisotopic method for analyzing receptor gene expression using reverse transcription-polymerase chain reaction (RT-PCR) and laser densitometry of agarose gels. Densitometry of known amounts of double stranded DNA molecular weight standards was used to estimate yield of glutamate receptor RT-PCR product (in ng DNA) obtained from rat brain. While unable to provide absolute quantitation of gene expression, this method using standard techniques allows for rapid analysis of relative levels of receptor gene expression without using radioactivity.

Abbott-81988 (A-81988) was selected from a series of related compounds as a highly potent and selective antagonist of angiotensin receptors. In the rabbit aorta, A-81988 exhibited a pA2 of 10.12 (+/- 0.08) vs. angiotensin-II, for type 1 receptors (AT1), and the antagonism appeared competitive. These results agreed with radioligand assays in which A-81988 inhibited the binding of [125I]-Sar1-Ile8-Angiotensin-II to rat liver membranes with a pKI of 9.12 (+/- 0.63). A-81988 was selective for AT1 receptors based on its lack of activity at other sites, such as aortic alpha 1 receptors. Moreover, A-81988 lacked affinity for AT2 receptors of bovine cerebellar membranes or for alpha or beta adrenergic receptors in binding assays. A-81988 lowered blood pressure significantly in vivo in renal artery-ligated rats at doses of 0.3 mg/kg administered either i.v. or p.o. The compound was rapidly and almost completely absorbed from the duodenum of anesthetized rats and demonstrated very low first-pass metabolism in the rat liver. These properties of selectivity toward and potency for antagonizing AT1 receptors, activity in lowering blood pressure in experimental animals, and favorable pharmacokinetic properties indicate that A-81988 should be a useful antihypertensive agent in man.

The development of the GABAA/Benzodiazepine receptor (GABAAR) in the red nucleus was studied using 3H-flunitrazepam (FNZ) as the probe. Saturation binding assay showed that the Bmax of the ligand to the membranes of the nucleus increased from 0.50 +/- 0.04 nmol/mg protein at postnatal day 4, to 0.71 +/- 0.1 and 0.78 +/- 0.08 at day 7 and day 10. At day 20 the Bmax decreased to a level near day 4 and persisted until day 40. However, the affinity of 3H-FNZ to the receptor remained quite constant. At least 4 proteins of 51kD, 53kD, 59kD and 62kD in the nucleus were labeled by 3H-FNZ, as revealed from photoaffinity binding and SDS-PAGE. The labeling of 53kD, 59kD and 62kD was high at earlier ages than day 10, whereas the 51kD was predominent from day 10 to day 40. Receptor binding autoradiography of the nucleus also showed that the most dense labeling was seen around day 10. The early transient increase in the GABAAR of the red nucleus may indicate the plasticity of the nucleus in response to environmental changes after birth.

Multiple types of dopamine D2-like receptors (D2, D3, D4) have been identified. Differences in pharmacology among these receptors may have profound clinical ramifications for the treatment of psychosis. Analysis of the structure and function of their binding sites requires a source of large amounts of receptor, uncontaminated by the other types of D2-like receptor. We engineered a recombinant baculovirus containing the human D2 receptor cDNA (DRD2) to express this receptor in insect cells. Spodoptera frugiperda cells (Sf9 and Sf21) and Trichoplusia ni cells (TN-5) were infected with the recombinant baculovirus. Binding of the D2 antagonist [3H]YM-09151-2 to membranes fractions of these cells peaked at a specific activity of 5-8 pmol/mg protein, approximately 40 times that of membranes from bovine striatum. The receptor expressed in Sf9 cells was similar to that of striatum in its affinities for D2 agonists and antagonists. Sodium ion stimulated [3H]YM-09151-2 binding to D2 receptor in infected Sf9 cell membranes. This effect was fit by an allosteric model which predicted the apparent affinity of [3H]YM-09151-2. The D2 receptor expressed in Sf9 and TN-5 cells was photolabeled with N-(p-azido-m-[125I]iodophenylethyl)spiperone. The specifically labeled component(s) ran as a broad band of apparent molecular weight between 54,000 and 60,000. Deglycosylation of the labeled component(s) reduced its molecular weight to 46,000.