Biomedical subjects

A M Traish

Publications and source records attributed to A M Traish.

At least 55 records · Page 3Linked to original sources

PubMed1995

Expression of functional muscarinic acetylcholine receptor subtypes in human corpus cavernosum and in cultured smooth muscle cells.

Relaxation of the trabecular smooth muscle, which is necessary for penile erection, is controlled locally by neurotransmitters and vasoactive agents. The goal of this study was to identify and characterize muscarinic acetylcholine receptor (mAChR) subtypes expressed in cultured human corpus cavernosum smooth muscle cells (HCC SMC). Binding analysis with L-[benzilic-4,4'-3H(N)]quinuclidinyl benzilate ([3H]QNB) demonstrated the expression of specific muscarinic receptor binding sites in HCC SMC. Analysis of total RNA isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated the expression of mRNA transcripts for m1, m2, m3, and m4 mAChR subtypes in whole tissue and m2 and m4 subtypes in cultured cells. In situ hybridization with specific m2 and m4 probes further confirmed the expression of m2 and m4 mRNA transcripts in cultured cells. Carbachol (CCh), a nonselective cholinergic agonist, inhibited cAMP synthesis at low concentrations (0.1-1 microM) and stimulated cAMP synthesis at high concentrations (100 microM), in cultured HCC SMC. CCh (100 microM) further augmented forskolin (FSK), isoproterenol (ISO), and prostaglandin E1 (PGE1)-induced cAMP synthesis. These observations suggest that, in vivo, in HCC, ACh may activate m3 mAChR subtypes on endothelial cells or m2 and m4 subtypes on the SMC. Although m2 and m4 are thought to inhibit adenylate cyclase (AC), the augmentation of cAMP synthesis by high concentrations of CCh in SMC suggests an alternative mechanism of coupling to G-proteins that stimulates AC activity. These studies show that HCC tissue expresses different subtypes of mAChR (m1, m2, m3, and m4), whereas cultured HCC SMC express m2 and m4 subtypes. It is suggested that m2 and m4 receptor subtypes may play an important role in maintaining trabecular smooth muscle tone in vivo. The augmentation of FSK-, ISO, and PGE1-induced cAMP synthesis by CCh suggests possible development of a multidrug therapeutic approach to treatment of erectile dysfunction.

Adenylyl Cyclases↗

PubMed1994

Detection of m2 muscarinic acetylcholine receptor mRNA in human corpus cavernosum by in-situ hybridization.

Relaxation and contraction of the smooth muscle of human corpus cavernosum (HCC) of the penis is essential for penile erection and detumescence. Relaxation of the smooth muscle is controlled, locally, by cholinergic, adrenergic and nonadrenergic, noncholinergic neurotransmitters as well as by the vascular endothelium, which lines the lacunar spaces and releases endothelium-derived relaxing factor. Cholinergic neurotransmitters are postulated to act on other neuroeffectors and on the endothelium rather than act directly on the trabecular smooth muscle. We use in-situ hybridization to determine the presence and distribution of muscarinic acetylcholine receptor (mAChR) subtype mRNA in HCC. First, we verified that the riboprobe used for in-situ hybridization is specific for m2 mAChR subtype using Chinese Hamster Ovary cells transfected with m2 mAChR subtype. Then, we validated that the tissue obtained from surgical specimens is adequate for in-situ hybridization. The data demonstrate that m2 mAChR subtype mRNA is expressed in HCC smooth muscle cells. m2 mAChR subtype mRNA expression is not detected in endothelium or nerves. It is possible that this receptor subtype plays a role in smooth muscle contraction.

Gene Expression↗

PubMed1994

7 alpha-17 alpha-Dimethyl-19-nortestosterone (mibolerone) induces conformational changes in progesterone receptors distinct from those induced by ORG 2058.

Using synthetic peptides with sequences derived from specific regions of human estrogen (ER) and progesterone (PR) receptors, we have developed site-directed monoclonal and polyclonal antibodies to specific domains of these receptors. These antibodies interact specifically with the native (nondenatured) receptors and detect changes in the conformation of these proteins. Monoclonal antibody PR-AT 4.14 bound more tightly to PR-ORG 2058 complexes than to PR-7 alpha,17 alpha-dimethyl-19-nor-testosterone (7 alpha,17 alpha, DMNT; mibolerone) complexes, suggesting possible ligand-induced conformational changes in PR. In the absence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 4S-5S entity on sucrose density gradients (SDG) containing 0.4 M KCl. In the presence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 7-8S complexes. In contrast, at the same concentrations of antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes sedimented at 4S-5S region in the absence of the antibody and as two populations in the presence of the antibody, suggesting that the antibody did not recognize all of the PR-7 alpha,17 alpha, DMNT complexes. To exclude the possibility that the inability of the antibody to recognize receptor-[3H]7 alpha,17 alpha, DMNT complexes was due to its binding to androgen receptors, unlabeled 5 alpha-dihydrotestosterone (5 alpha-DHT) (50 nM) was added to the incubation to inhibit 7 alpha,17 alpha, DMNT binding to androgen receptors. While PR-[3H]ORG 2058 complexes were immunoprecipitated in the presence of the antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes were only partially immunoprecipitated, further confirming the results obtained with SDG.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Immuno-electron microscopic localization of estradiol receptor in cells of male and female reproductive and non-reproductive organs.

The localization of estradiol receptor (ER) in various tissues and their distribution in sub-cellular compartments were studied by means of immunogold-electron microscopic methods using a site-directed polyclonal antibody developed against a peptide from the DNA binding site of ER. This method was used to determine the presence and localization of ER in tissues and cells of male and female reproductive and non-reproductive organs. In the female reproductive tract, endometrial cells and the cells of the corpus luteum were found to contain ER. In non-reproductive organs of both sexes the following cell types showed significant labeling: hepatocytes, epithelial duodenal cells, striated muscle fibers, cells of the proximal convoluted tubules of the kidney, lymphocytes, neurons, and adipose cells. Alveolar epithelial cells were studied only in female specimens and were labeled by the anti-ER. Prostatic and epididymal epithelial cells were found to be labeled in the male reproductive organs. In all these cells a higher density of label was found in the nucleus, especially in the space between the clumps of compact chromatin, as was previously found in epithelial endometrial cells. These results suggest that estradiol exerts its effects through a common nuclear mechanism in cells of male and female reproductive and non-reproductive organs.

Animals↗

PubMed1994

Characterization of muscarinic acetylcholine receptors in cultured bovine aortic endothelial cells.

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.

Animals↗

PubMed1994

Characterization of human retinoic acid receptor alpha 1 expressed in recombinant baculovirus-infected Sf9 insect cells.

Full-length human retinoic acid receptor alpha 1 (hRAR alpha 1) was expressed in Sf9 insect cells using the baculovirus expression vector system (BEVS). Western blot analysis using a specific anti-RAR peptide antiserum detected two major protein bands with apparent mol wts of approximately 54 and approximately 51 kDa in extracts from insect cells infected with recombinant hRAR alpha 1 Autographica californica (AcNPV) baculovirus. Analysis of recombinant extracts from Sf9 cells labeled in vivo with [32P]orthophosphate suggested that the recombinant protein was phosphorylated. A component in the recombinant nuclear extracts specifically bound [3H]all-trans-retinoic acid (RA) and sedimented in sucrose density gradient centrifugation as a single, symmetric peak with a sedimentation coefficient of approximately 3.6S, corresponding to a protein of approx 50 kDa. Scatchard analyses determined that [3H]RA was bound in recombinant extracts by a single class of binding sites with an apparent dissociation constant of approximately 0.3 nM and nuclear and cytoplasmic extracts contained approximately 1200 and approximately 200 pmoles, respectively, of unoccupied receptor per mg protein. In competitive ligand binding assays, relative binding affinities of 9-cis- and 13-cis-RA for hRAR alpha 1 in nuclear extracts were about threefold and sixfold lower than all-trans-RA, whereas all-trans-retinol, -retinaldehyde, and -retinyl acetate demonstrated relatively weak binding. In gel mobility shift assays, the electrophoretic migration of a [32P]-labeled oligonucleotide containing the retinoic acid response element of the RAR beta gene was retarded in the presence of recombinant nuclear and cytoplasmic extracts. The apparent complex formation between recombinant hRAR alpha 1 and beta RARE was greatly enhanced by the addition of nuclear extract from wild-type AcNPV-infected Sf9 cells, possibly because of heterodimer formation between recombinant hRAR alpha 1 and a metazoan RXR homolog. Thus, recombinant hRAR alpha 1 expressed at high levels in Sf9 insect cells exhibited biochemical properties of the native protein, including nuclear translocation, specific high affinity ligand and RARE binding, and possible heterodimer formation.

Amino Acid Sequence↗

PubMed1992

Endothelin in the urinary bladder. I. Synthesis of endothelin-1 by epithelia, muscle and fibroblasts suggests autocrine and paracrine cellular regulation.

The synthesis and localization of endothelin-1 were studied in human and rabbit bladder. In addition, the effects of endothelin-1 on smooth muscle tone and cholinergic neurotransmission were investigated in rabbit bladder. Endothelin-like immunoreactivity was localized in the transitional epithelium, serosal mesothelium, and vascular endothelium; smooth muscle of the bladder (non-vascular) and that of blood vessels; and fibroblasts. With in situ hybridization, transcripts of endothelin messenger ribonucleic acid (mRNA) were localized with the same cellular distribution as endothelin-like immunoreactivity, in bladder tissue. Northern blot analysis of bladder RNA confirmed the expression of preproendothelin-1 mRNA. Rabbit bladder strips in organ chambers contracted when exposed to endothelin-1 and this response was partially attenuated by calcium channel blockers or by removal of extracellular calcium. Transmural electrical stimulation of rabbit bladder strips elicited contractions that were greatly reduced by atropine. The remaining atropine resistant component was blocked by alpha, beta-methylene ATP, which desensitizes purinergic receptors. Endothelin-1 caused a small but consistent attenuation of the atropine sensitive component of the neurogenic contraction, while it had no effect on the atropine resistant component. The localization of endothelin synthesis in epithelia, smooth muscle, and fibroblasts suggests that endothelin may act as an autocrine hormone in the regulation of the bladder wall structure and smooth muscle tone. In addition, endothelin-1 may regulate cholinergic neurotransmission by a paracrine mechanism.

Animals↗

PubMed1992

Monoclonal antibodies to human endothelin-1: characterization and utilization in radioimmunoassay and immunocytochemistry.

A host of monoclonal antibodies directed against human endothelin-1 (ET-1) has been developed and characterized. The antibodies reacted with ET-1 specifically and with high affinity, as determined by competition analysis and sucrose density gradients. The antibodies did not cross-react with neuropeptide YY, beta-endorphin, calcitonin gene-related peptide, secretin or somatostatin. The antibodies cross-reacted with big endothelin (B-ET), endothelin-2 (ET-2), vasointestinal constrictor peptide (VIC), and endothelin-3 (ET-3) albeit with varying affinity but did not cross-react with sarafotoxin (SRTX-6b). None of the antibodies reacted with the C-terminal hexapeptide (HXPT) of ET-1, indicating that the epitopes are not located within this region of ET-1. The monoclonal antibodies exhibited binding activity in dilutions ranging from 1:1000, to 1:10(6). The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG gamma 1, two were IgM and one of the IgG gamma 2a subclass. The antibodies detected immunoreactive ETs by radioimmunoassay and in immunocytochemical localization, suggesting the potential use of these antibodies as tools to determine the concentration of ETs in biological fluids and in immunocytochemical localization of ETs in specific cell types in various tissues.

Amino Acid Sequence↗

PubMed1991

Immunoelectron microscopic localization of estrogen receptor on pre-mRNA containing constituents of rat uterine cell nuclei.

The localization and quantitative changes of estradiol receptor (ER) were studied by means of immunogold-electron microscope methods using a polyclonal antibody directed against an amino acid sequence representing the DNA binding site of ER, a monoclonal antibody against hnRNP core protein, and anti-DNA antibody. The uteri of normal rats in estrus and those of ovariectomized females were used. Ovariectomized rats were studied 21 days after surgery at different times after the injection of normal saline or estradiol-17 beta. The density of labeling was measured in interchromatin space, compact chromatin, nucleolus, cytoplasm, and background of epithelial cells, muscle cells, and fibroblasts. In the three types of cells ER was found mainly on extranucleolar ribonucleoprotein (RNP) fibrils. In epithelial and muscle cells the nucleolus was labeled but compact chromatin was not labeled. In epithelial cells there was a low but significant labeling of the cytoplasm. Fibroblasts exhibited a low labeling of the compact chromatin. Ovariectomy did not change these distributions. The estradiol injection increased labeling in all compartments of epithelial and muscle cells but decreased the labeling of compact chromatin of fibroblasts. These results show: (a) that ER is mainly nuclear but it is also present in the cytoplasm, (b) that ER binds to the nuclear particles containing newly synthesized RNA, and (c) that the binding to RNPs does not block the DNA binding domain of the ER.

Animals↗

PubMed1991

Endothelin: localization, synthesis, activity, and receptor types in human penile corpus cavernosum.

The localization, synthesis, and activity of endothelin and the receptor types mediating its effects in penile corpus cavernosum were investigated in whole tissue and in cultured cells derived from this tissue. With immunocytochemistry, utilizing an antiendothelin 1 (ET-1) monoclonal antibody, endothelin-like immunoreactivity was localized intensely in the endothelium and to a lesser degree in the trabecular smooth muscle. Human corpus cavernosum endothelial cells in culture expressed preproendothelin 1 mRNA, as determined by Northern blot analysis. Significant amounts of endothelin-like immunoreactivity were measured by radioimmunoassay in the supernatants of corpus cavernosum endothelial cells in culture. Endothelins are potent constrictors and caused long-lasting contractions of corporeal strips in organ chambers. Equilibrium binding analysis of endothelins to their receptor sites revealed high-affinity, specific, and saturable binding of labeled endothelins to corporeal membranes. Competition binding experiments demonstrated receptors with high affinity for ET-1 and -2 and low affinity for ET-3 and another, less abundant, set of receptors with high affinity for ET-1, -2, and -3. Affinity labeling of endothelins to corporeal membranes, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, revealed that ET-1 and -2 cross-linked specifically to three different molecular mass components (75, 52, and 34 kDa). ET-3 bound only to the 34-kDa component. It is concluded that human corpus cavernosum endothelium has the ability to synthesize and release endothelin, that endothelins contract corporeal smooth muscle, and that at least two distinct endothelin receptors may exist and are differentiated by their affinity for ET-3.

Affinity Labels↗

PubMed1991

Arginine vasopressin stimulates human mesangial cell production of endothelin.

Endothelin (ET) is a vasoactive peptide produced by both endothelial epithelial cells with documented mitogenic action on mesangial cells. The present studies were designed to test the hypothesis that ET is also produced by human mesangial cells (HMC) and that other mitogens such as arginine vasopressin (AVP) and insulin stimulate cellular proliferation, in part, through modulation of endogenous production of this peptide. Studies were conducted on cultured normal HMC between the third and seventh passages. All mitogenesis experiments were carried out in 96-well plates and assessed by tritiated thymidine incorporation into DNA under various concentrations of AVP in the presence and absence of insulin, antiendothelin antisera (ETAS), a MAb against ET-1 (AbET), and a vasopressin-1 receptor antagonist. ET concentrations were measured daily from conditioned medium by a sensitive and specific RIA. ET was present in all concentrations of FCS as well as conditioned medium compared with medium alone. AVP (10(-6) M) in the presence of insulin increased ET production by quiescent HMC by 261% as well as cellular proliferation by 440% after 48 h incubation. In addition, cells cultured with ETAS or AbET demonstrated a blunted mitogenic response to AVP, a response not observed in cells cultured with ETAS where ET was added. Insulin significantly potentiated the mitogenic effects of AVP as well as media levels of ET, an effect significantly blunted by AbET. We conclude that ET is produced by HMC and its production is affected, in part, by both AVP and insulin. ET may thus serve to modulate the mitogenic effects of AVP on human mesangial cells.

Arginine Vasopressin↗

PubMed1991

Physicochemical characterization and solubilization of endothelin receptors.

[125I]Endothelin-1 (ET-1) bound to specific endothelin receptors (ET-R) with high affinity (Kd = 0.4-1 nM) and dissociated very slowly from ET-R at 37 degrees C (K-1 = 2.4 x 10(-3)/h). The binding of ET-1 was reduced in acidic (pH = 4) and alkaline (pH > 8.5) medium but was stable at near neutral pH (pH 6.5-7.5). Covalent affinity labeling of ET-R with [125I] endothelins (ET-1,2,3) demonstrated that ET-1 and ET-2 bound specifically to three proteins with approximate mol masses of 75, 52, and 34 kDa, whereas [125I]ET-3 bound mainly to a protein with a mol mass of 34 kDa. The binding of [125I]ET-1 to the three mol-mass species was effectively displaced with ET-1 and ET-2. However, ET-3 displaced the binding to the 34-kDa band, only. ET-R were solubilized, in active endothelin-binding forms, with 1% digitonin and 0.1% cholate. Solubilized ET-R sedimented on sucrose density gradients (SDG) containing 0.1% CHAPS, 10% glycerol, and 0.4 M KCl, as 7-8S complexes. Binding of ET-1, ET-3, vasoactive intestinal constrictor (VIC) or sarafotoxin (SRTX-6b) to the solubilized ET-R, and subsequent analysis on SDG, demonstrated similar sedimentation characteristics, suggesting that these peptides are bound to similar receptors.

Affinity Labels↗

PubMed1990

Development and characterization of monoclonal antibodies to a specific domain of human estrogen receptor.

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201-215, 231-245, and 247-261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247-261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [3H]estradiol. The antibodies bound to ERs with high affinity (KD = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1,000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens.

Amino Acid Sequence↗

PubMed1990

Site-directed polyclonal antibodies inhibit binding of activated estrogen receptor to DNA.

We have generated three polyclonal antisera to the DNA-binding domain of the human estrogen receptor (hER). Antiserum AT2A was generated against a peptide spanning amino acids 231-245 of hER, while antisera AT3A and AT3B were generated against a peptide spanning amino acids 247-261 of hER. The interaction of these three antisera with ER has been characterized by sucrose density gradient analysis. The antisera bound to the unactivated (8S), salt-activated (4S), and heat transformed (5S) ER complex. All the antisera appeared to be site-specific since binding of salt-activated ER to the antisera was blocked by the presence of excess free synthetic peptides. Antisera AT3A and AT3B inhibited the binding to DNA of the KCl-activated 4S ER and the heat-transformed 5S ER. Although antiserum AT2A bound to ER, it did not inhibit DNA binding of activated ER complexes. The ability of antisera AT3A and AT3B to inhibit ER binding to DNA was concentration dependent. Once bound to the DNA, ER complexes were not significantly affected by incubation with the antisera, suggesting that binding of DNA to ER inhibits antibody ER interaction and renders that domain inaccessible to the antibodies. These results demonstrate that site-directed antibodies to ER inhibit binding of activated ER complexes to DNA in vitro.

Antibodies↗

PubMed1990

Characterization of muscarinic acetylcholine receptors in human penile corpus cavernosum: studies on whole tissue and cultured endothelium.

The binding characteristics of the muscarinic acetylcholine receptor antagonist, [3H]quinuclidinyl benzilate, to isolated membranes of human corpus cavernosum and endothelial cells, cultured from this tissue, were investigated. [3H]quinuclidinyl benzilate bound to membranes of human corpus cavernosum and endothelial cells with high affinity and limited capacity. Analysis of the binding data by Scatchard plot revealed the presence of one class of binding sites. The ligand binding specificity was determined by competitive binding assay. The data obtained show that [3H]quinuclidinyl benzilate was displaced by unlabeled competitors in the following order of efficacy in both membrane preparations: quinuclidinyl benzilate greater than scopolamine greater than atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methiodide, M3 antagonist greater than pirenzepine, M1 antagonist greater than oxotremorine greater than (4-hydroxy-2-butynyl)trimethylammonium chloride m-chlorocarbanilate, M1 agonist greater than carbachol greater than hexamethonium. Solubilization of the muscarinic acetylcholine receptors from human corpus cavernosum and endothelial cells, with 1% digitonin and 0.02% cholate and subsequent analysis on sucrose density gradients, demonstrated the presence of a macromolecule specifically bound to [3H]quinuclidinyl benzilate sedimenting at the 6.2 S region of the gradient. These results demonstrate the presence of muscarinic acetylcholine receptors in human corpus cavernosum and in endothelial cells cultured from this tissue.

Binding Sites↗

PubMed1990

Monoclonal and polyclonal antibodies to human progesterone receptor peptide-(533-547) recognize a specific site in unactivated (8S) and activated (4S) progesterone receptor and distinguish between intact and proteolyzed receptors.

We have synthesized three peptides with amino acid sequences corresponding to amino acids 533-547, 597-611, and 765-779 of the human progesterone receptor (hPR). These peptides were conjugated to keyhole limpet hemocyanin and injected into mice and rabbits to develop antibodies to hPR. Antibodies to the undenatured form of PR were elicited only by the peptide with amino acid sequence 533-547. Fusion of SP2/0 myeloma cells with spleen cells from mice immunized with this peptide produced several active clones. Rabbit sera from immunized animals produced one antiserum that reacted with the undenatured form of PR. One monoclonal antibody (PR-AT 4.14) and one antiserum (PR-AT533) raised against peptide-(533-547) were characterized. Binding of these antibodies to the undenatured form of PR was demonstrated by analysis of the antibody-receptor complexes on sucrose density gradients and by immunoprecipitation techniques. Binding of PR to the antibodies was inhibited by excess peptide. The antibodies did not react with estrogen, glucocorticoid, or androgen receptors, but recognized PR from human breast cancer as well as calf, rabbit, mouse, and rat uteri, indicating that this epitope was conserved among these species. Based on sucrose density gradient analysis of PR prepared and labeled in the presence of proteolysis inhibitors and sodium molybdate, the antibodies bound to a site on the intact undenatured PR, but failed to bind to partially degraded steroid-binding form of the receptor, suggesting that the antibody-binding domain is at or near a site sensitive to proteolysis.

Amino Acid Sequence↗

PubMed1988

Validation of the exchange assay for the measurement of androgen receptors in human and dog prostates.

This study describes an exchange assay for measurement of cytosolic and nuclear androgen receptors (AR) in human and dog prostates. Efficient replacement of endogenously bound ligand from the receptor with [3H] mibolerone was achieved by incubation of cytosolic or nuclear fractions at 0 degree C for 72 h in the presence of 0.15M NaSCN and 15% sucrose. It was demonstrated that in the presence of the chaotropic salt rapid steroid dissociation took place at 0 degree C followed by [3H] mibolerone binding; sucrose protected the AR from denaturation by NaSCN. Combination of these two reagents, therefore, allowed quantitative androgen exchange at 0 degrees C. Receptors determined by this exchange procedure are specific for androgens, of high affinity (KD 2-5 nM), and sedimented on sucrose gradients as 4-4.6S entities. Use of [3H] mibolerone minimized interference from plasma proteins and reduced nonspecific binding. This exchange assay has now been applied to quantitation of cytosolic and nuclear AR in small tissue samples (50 mg). Thus it is possible to measure AR in tissue samples obtained by needle biopsies and attempt to correlate receptor values to clinical response of prostatic cancer patients.

Animals↗

PubMed1987

Stimulation of lysyl oxidase (EC 1.4.3.13) activity by testosterone and characterization of androgen receptors in cultured calf aorta smooth-muscle cells.

Previous studies have indicated a greater incidence of atherosclerotic cardiovascular disease in men than in women of child-bearing age, suggesting that vascular interactions with sex steroids may effect pathogenesis in these cases. In the present study, it was found that the presence of 10-100 nM-testosterone in the growth medium of calf aortic smooth-muscle cells in culture stimulates lysyl oxidase activity approx. 2.5-fold in the medium and 5.5-fold in the fraction bound to the cell layer. Androgen receptors were identified in these cultured smooth-muscle cells, and their properties were very similar to those in the cytosolic fraction of whole bovine aortic tissue. These receptors appeared to be specific for androgen, of high affinity (Kd = 0.4 nM) and of low capacity (9000 sites/cell). The present results indicate that the aortic smooth-muscle cell is a cellular target for androgens, and thus raise the possibility that the development of fibrotic arterial lesions involving the deposition of excess collagen may in part be regulated by androgen-mediated stimulation of collagen cross-linkage formation as catalysed by lysyl oxidase.

Amino Acid Oxidoreductases↗