Identification of arrestin-like proteins in a human megakaryocytic cell line (HEL cells).

Proteins of the arrestin family contribute to the regulation of G-protein-mediated signal transduction in a number of tissues, possibly by a desensitization of the appropriate receptor(s). In this study we demonstrate the presence of arrestin-related proteins in a megakaryoblast-like cell line (HEL). Mouse monoclonal and rabbit polyclonal antibodies were prepared against visual arrestin, against a synthetic peptide'GFLGELTSSEVATEVPFRLM' (a pathogenic sequence corresponding to residues 340 to 359 of human visual arrestin), and against the peptide 'VDTNLIEFDTNDDDIV' that represents an aminoacid sequence present in beta -arrestins 1 and 2 but absent from visual arrestin.

Mineralocorticoid receptor-mediated signaling regulates the ion gated sodium channel in vascular endothelial cells and requires an intact cytoskeleton.

The PCR analysis followed by sequence alignment showed that both the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes were expressed in the human vascular endothelial cell line (ECV). The growth and multiplication of the ECV in culture were influenced by both aldosterone and the MCR-specific antagonist ZK 91587. Following double labelled immunofluorescence recorded by confocal microscopy, both the MCR and the ENaC were found to colocalize with the tubulin filaments in ECV cells in situ; no association was observed with cellular actin.

Paradoxical effects of mineralocorticoids on the ion gated sodium channel in embryologically diverse cells.

PCR analysis and Western blotting revealed the expression of the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes at the level of RNA, DNA, and protein in several leukemic cell lines, fibroblasts from human cornea, and epithelial cells from ocular tissues. Following immunofluorescence, the MCR appeared to be primarily nuclear whereas the ENaC was almost exclusively membrane-bound. Paradoxically, the MCR-specific antagonist ZK 91587 actually stimulated the multiplication of human erythroblastic leukemia cells, contrary to the inhibitory effect of the antagonist RU 26752 on the multiplication of corneal fibroblasts; both effects were opposed by aldosterone.

Immunochemical analysis of the sodium channel in rodent and human eye.

The presence of the amiloride-sensitive sodium channel (ASSC) in ocular tissues was studied with the aid of a polyclonal antiserum raised against the 14 amino acid peptide QGLGKGDKREEQGL. This sequence corresponds to the region 44-58 of the alpha subunit of the channel, termed ENaC, cloned from rat colon. The antibody titers, measured by the ELISA technique, rose to 1∶2560 4 weeks after immunization, and this bleed was used in all subsequent experiments.

Enhanced activation of the mineralocorticoid receptor in genetically hypertensive rats.

The relative abundance and availability of the mineralocorticoid receptor (MCR) appeared to be similar in the heart, kidney and ocular tissues of the genetically hypertensive SHR and normotensive WKY rats by a number of criteria including Western blotting, immunoprecipitation, dot blot analysis, and immunohistochemistry. On the other hand, the activation of the MCR, as judged by binding to DNA cellulose, was significantly enhanced in the hearts and kidneys of 14 week-old, hypertensive, SHR rats compared to the normotensive WKY animals. The activation of the renal MCR was elevated in the SHR strain even at the age of six weeks when the tail arterial pressure was statistically identical to that of the WKY strain.

Immunochemical demonstration of the mineralocorticoid receptor in ocular tissues.

We studied the presence of the mineralocorticoid receptor (MCR) in the eye with the aid of a number of immunochemical techniques. Immunoblotting with a polyclonal antibody, directed against the rat renal MCR, revealed a single band of about 102 kD in extracts prepared from whole bovine or rat retina similar to that observed in cytosol from the kidney and myocardium from these species. Isolated cells of the bovine retinal pigment epithelium (RPE) similarly exhibited a 98- to 102-kD band in Western blots developed with the aid of anti-MCR antiserum.

Evidence for receptor-mediated mineralocorticoid action in rat osteoblastic cells.

Aldosterone significantly enhanced the proliferation of osteoblastic cells from rat calvaria, and this effect was inhibited by RU 26752 and ZK 91587, two antagonists specific to the mineralocorticoid receptor (MCR). In addition, aldosterone inhibited the activity of alkaline phosphatase, a marker of the osteoblastic phenotype, and this effect was also reversed by RU 26752. Cytoplasmic staining of MCR was observed in rat calvaria osteoblasts incubated with a specific polyclonal antiserum raised against rat kidney MCR.

The mineralocorticoid hormone receptor and action in the eye.

Immunoblotting with a polyclonal antibody, directed against the mineralocorticoid receptor protein purified from rat kidney in presence of the receptor-specific ligand RU 26752, labeled a single 98-102 kDa band in soluble extracts from bovine retina and from cultured bovine retinal pigment epithelial cells, identical to the receptor in several other tissues from the rat. The antibody also immunoprecipitated the receptor-3H-RU 26752 complex in bovine retinal extract. The growth of the isolated pigment epithelial cells was inhibited by RU 26752 and ZK91587, two ligands specific to the mineralocorticoid receptor.

The antiglucocorticoid action of mifepristone.

Glucocorticoid hormones influence the physiological activity of almost all cell types in the mammal. This is accomplished via a soluble receptor that, in the presence of an appropriate steroid, modifies the activity of RNA polymerase by binding to the site where different factors assemble for the initiation of cell transcription. The development of antiglucocorticoids has permitted the molecular elucidation of a number of underlying events.

Properties of the mineralocorticoid receptor immunopurified from bovine kidney.

The mineralocorticoid receptor (MCR) from bovine kidney was purified on an affinity column containing covalently linked polyclonal IgG raised in the rabbit against rat kidney protein purified in the presence of RU 26752 that is specific to the MCR. The immuno-affinity eluate was excluded as a single peak during gel permeation chromatography and could be resolved as a single band of approximately 98 kDa by western blot and gel electrophoresis. Immunohistochemistry revealed MCR-specific staining in both the cortical and glomerular regions of bovine kidney.

Nicotiana tabacum contains a putative mineralocorticoid receptor.

Total protein from N. tabacum cell cytosol, partially purified by ammonium sulphate precipitation, contains a 52 kDa protein that tests positive for mineralocorticoid receptor (MCR)-like activity by Western blots, immuneprecipitation and photochemical cross-linking. Binding of RU 26752, a ligand specific to the MCR, suggests 100 fmol steroid bound per mg of tobacco protein; this is equivalent to about 6000 sites per cell assuming a 1/1 stoichiometry.

Analysis of steroid receptor domains with the aid of antihormones.

1. The receptors for steroid hormones consist of well defined domains with overlapping functions. 2.

Steroid receptor domain conformations and hormone antagonism.

Receptor stabilization, activation, dimerization, and binding to cognate sequences on DNA are possible with antagonists. Tissue-, steroid-, and species-dependent differences in all these parameters, despite identical structure of the receptor from various sources for any one steroid hormone class, suggest posttranslational modifications of a primary gene product. Clinically, it is now possible to visualize receptor-specific antihormone therapy of various steroid-dependent maladies (cancer of the breast, uterus, or prostate, Cushing's disease, hypertensive disorders, etc.

Immunochemical detection of the mineralocorticoid receptor in rat brain.

The mineralocorticoid receptor (MCR) in the brain of adult male rats was analyzed with the aid of an antiserum generated by immunizing rabbits with this protein purified biochemically from rat kidney. In Western blots, the antibody recognized a single band of protein of about 98 kD from all target tissues studied to date. The granular cells in the cerebellum appeared to be the richest region of the central nervous system in the MCR analyzed by dot blots and by immunoperoxidase staining.

Receptors for mammalian steroid hormones in microbes and plants.

Steroids are of universal occurrence, present variously as cell wall constituents and bioregulators. A number of bacteria, fungi, and photosynthetic vascular plants synthesize steroids that are hormonally active in the animal world. The cellular effect of such steroids in microbes and plants appears by and large to be comparable to that in mammals.

[The Cordeliers University Center. A short guide for a visitor].

The history of the Pierre and Marie Curie University Cordeliers Center is summarized. A few traces of the history of this part of the latin quarter still remain. The oldest souvenir is the plain but impressive Refectory of the Cordeliers.

Receptor mediated mineralocorticoid action in alga cell mutants.

The multiplication of Chlamydomonas cells can be arrested by the spirolactone derivative RU 26752 and this is fully reversible by the natural hormone aldosterone. Continuous growth in the presence of RU 26752 led to the isolation of a population subsequently resistant to the action of mineralocortoid analogues, due possibly to the selection of mutant cells. Immunophotochemical evidence is provided for a 52 kDa protein that possesses functional steroid and DNA binding domains.

Mineralocorticoid hormone action in plant cells.

The multiplication of Chlamydomonas reinhardtii wild type cells can be arrested by the spirolactone RU 26752 and this is fully reversible by the natural mineralocorticoid aldosterone. Evidence is presented for a 52 kDa protein that possesses functional DNA and ligand binding domains and tests positive for mineralocorticoid receptor-like activity by immuneprecipitation, macroaggregation, and photoaffinity. The regulation of trans-activation by steroid hormones in the animal world would therefore appear to be just as valid for the plant kingdom, thereby providing a new model for genetic analysis.

Immunophotochemical analysis of mineralocortin by polyclonal antibodies against the native receptor from rat kidney.

We have obtained a polyclonal antiserum by immunizing fawn Burgundy rabbits with the mineralocorticoid receptor (MCR) purified biochemically from rat kidneys. High titers of anti-MCR activity were obtained in radioimmunoassays within 3 weeks and increased with a booster shot. In Western blot analysis, the antibody revealed a major band of 94-98 kDa in renal cytosol from rat and beef kidneys.

Generation of polyclonal antibodies against the mineralocorticoid receptor and analysis of mineralocortin in rat myocardium by immunophotochemistry.

Fawn, Burgundy rabbits were immunized with the mineralocorticoid receptor (MCR) purified biochemically from rat kidney by a simple, two step procedure. High anti-MCR titers were observed in radioimmunoassays just 3 weeks after the initial injection and increased further with time. Western blot analysis revealed a single band of 94-98 kDa in renal and cardiac cytosol from the rat, like the antigen prepared biochemically.

Rat lung possesses the mineralocorticoid receptor.

Lung cytosol from male, adrenalectomized rats was screened for the mineralocorticoid receptor (MCR) by a polyclonal antiserum raised in the rabbit against rat renal antigen. Western blot analysis revealed a single 98 kDa band, like the MCR purified biochemically. The MCR could also be photolabelled for the first time by 3H-R 5020 in this very 98 kDa region that was displaced by RU 26752 specific to MCR.

Evolving trends in steroid hormone receptor research.

Recent advances have challenged classical notions regarding the nature of steroid hormone receptors in the cell including localization, activation, configuration, and stability. Molecular biology has revealed a remarkable similarity in the primary structure of a wide variety of receptor classes that goes beyond steroid action. Post-translational modification of a primary unit, expressed in response to genetic conservatism, would appear to assure receptor dynamics specific to hormone-, organ-, and tissue-dependent processes, and may even lead to toxicity and oncogenicity.

Purification and characterization of the activated mineralocorticoid receptor from rat kidney.

1. The mineralcorticoid receptor (MR) from rat kidney was purified within 8 hr by the following, successive steps: stabilization with synthetic, tritiated steroids (RU 26752 or R 5020), phosphocellulose passage, heat activation (25 degrees C), and DNA-cellulose batch elution. 2.

Analysis of the mineralocorticoid receptor in rat heart with the aid of two new spirolactone derivatives.

Two derivatives of spirolactone, synthesized in an effort to eliminate the obnoxious side effects of the native molecule, were employed to dissect various aspects of the MR structure and function in rat heart. The introduction of a propyl residue in position 7 of spirolactone produced a molecule (RU 26752) that exhibited an increased affinity for the agonist specific MR, and furthermore revealed an antagonist-specific MR population in the target organ heart but absent from nontarget lung and liver. The specificity for both sites increased when a methoxycarbonyl group was introduced in the 7 position (ZK 91587).