Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland.: Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)51[Tyr(Me)2, Thr4,Tyr-NH29]OVT

1989 ◽  
Vol 67 (2-3) ◽  
pp. 152-162 ◽  
Author(s):  
Melvyn S. Soloff ◽  
Mats A. Fernström ◽  
Martha J. Fernström
Keyword(s):  
Mammary Gland ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Inositol Phosphate ◽  
Mammary Tissue ◽  
Selective Antagonist ◽  
High Affinity ◽  
Oxytocin Receptors ◽  
Rat Mammary Gland ◽  
Stimulation Of

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)51[Tyr(Me)2,Thr4,Tyr-NH29]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)51[Tyr(Me)2,Thr4,Tyr-NH29]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding. The high affinity, specificity, and specific activity of the iodinated antagonist should make it very useful in further studies to discriminate between oxytocin and AVP receptors, demonstrate oxytocin receptors with small amounts of samples, perform autoradiographic studies with short-term exposures, and to purify oxytocin receptors.Key words: oxytocin, vasopressin, receptor, mammary gland, antagonist.

[3H]-[Thr4,Gly7]OT: a highly selective ligand for central and peripheral OT receptors

1988 ◽  
Vol 254 (1) ◽  
pp. E31-E38 ◽  
Author(s):  
J. Elands ◽  
C. Barberis ◽  
S. Jard
Keyword(s):  
Mammary Gland ◽  
Arginine Vasopressin ◽  
Plasma Membranes ◽  
Dissociation Constants ◽  
Synaptic Plasma Membranes ◽  
High Affinity ◽  
Receptor Sites ◽  
Oxytocin Receptors ◽  
Selective Ligand ◽  
Equilibrium Dissociation Constants

Oxytocin receptors in rat hippocampal synaptic plasma membranes were compared with mammary gland and uterine oxytocin receptors. For this purpose, a highly specific oxytocic agonist [Thr4,Gly7]oxytocin was tritiated. We demonstrated that this ligand labels oxytocin receptors selectively. Scatchard analyses revealed a high affinity for all the oxytocin receptors investigated, with equilibrium dissociation constants between 1.0 and 2.0 nM. Binding appeared to take place at a single population of receptor sites. Competition experiments confirmed the high affinity of arginine vasopressin for hippocampal oxytocin receptors but also revealed that mammary gland and uterine oxytocin receptors do not discriminate more efficiently between oxytocin and arginine vasopressin. This lack in specificity is not affected by applying different concentrations of Mg ions.

scholarly journals Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding

1982 ◽  
Vol 203 (1) ◽  
pp. 45-50 ◽  
Author(s):  
P M Ahmad ◽  
D S Feltman ◽  
F Ahmad
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Specific Activity ◽  
Simple Procedure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Performic Acid ◽  
Mammary Tissue ◽  
Rat Mammary Gland

A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity, with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 480 000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4′-phosphopantetheine in the native enzyme. Rat mammary-gland fatty acid synthase required free CoA for activity.

Anti-TSH receptor antibodies in Graves' disease modulate the kinetic behaviour of autologous thyroid TSH receptors. Evidence of the IgG-induced cross-linkage of autologous TSH receptors

1984 ◽  
Vol 105 (3) ◽  
pp. 330-340 ◽  
Author(s):  
Tjerk W. A. de Bruin ◽  
Daan van der Heide ◽  
Maria C. Krol
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Graves Disease ◽  
Kinetic Behaviour ◽  
High Affinity ◽  
Cross Linkage ◽  
Receptor Antibodies ◽  
Tsh Receptor Antibodies

Abstract. The effect of the anti-TSH receptor antibodies present in the sera of 8 patients with Graves' disease on the affinity constant (Ka) and the number (R) of TSH receptors in autologous human thyroid plasma membranes was investigated. Kinetic analysis of [125I]bTSH binding to human thyroid plasma membranes in the presence of autologous Graves' and normal gammaglobulins was carried out by means of a computer fitting programme. Analysis of the TSH-TSH receptor interaction in the presence of TSH alone yielded curvilinear Scatchard plots, indicating the existence of two independent classes of binding sites (high affinity Ka: 8.5 ± 4.8 × 108 m−1; low affinity Ka: 5.3 ± 2.7 × 106 m−1). Similarly the Scatchard plot for this interaction in the presence of normal gammaglobulins is also curvilinear. Linear Scatchard plots, indicating the existence of only one class of high affinity TSH binding sites (Ka: 3.5 ± 1.8 × 108 m−1), were obtained for both autologous gammaglobulins and pure IgG from 8 patients with Graves' disease. The number of high affinity TSH binding sites in the presence of Graves' gammaglobulins had increased on the average by a factor 3.76 ± 0.74 (sd) with respect to the number found in the presence of normal gammaglobulins. This marked change in the kinetic behaviour of the TSH binding sites provided evidence that there is a direct interaction between anti-TSH receptor antibodies and autologous TSH receptors. Divalency of Graves' IgG or linkage of Fab fragments by anti-Fab antiserum proved to be necessary to produce this specific change in the kinetic behaviour of TSH binding sites. Graves' IgG monovalent Fab and Fc fragments had no effect. We suggest that the mechanism by which anti-TSH receptor antibodies in Graves' disease mimick the biological action of TSH is the IgG-induced cross-linkage of TSH receptors.

Activation of endothelin ETB receptors increases glomerular cGMP via an L-arginine-dependent pathway

1992 ◽  
Vol 263 (6) ◽  
pp. F1020-F1025 ◽  
Author(s):  
R. M. Edwards ◽  
M. Pullen ◽  
P. Nambi
Keyword(s):  
Nitric Oxide ◽  
Guanylate Cyclase ◽  
Binding Sites ◽  
Soluble Guanylate Cyclase ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
High Affinity ◽  
Dependent Pathway ◽  
Stimulation Of

The effects of endothelins (ET) on guanosine 3',5'-cyclic monophosphate (cGMP) levels in intact rat glomeruli were examined. ET-3 produced a rapid approximately fivefold increase in cGMP levels with the maximum effect occurring at 1 min. The ET-3-induced increase in cGMP accumulation occurred in the absence and presence of 3-isobutyl-1-methylxanthine. ET-1, ET-2, ET-3, and the structurally related toxin, sarafotoxin S6c, all increased glomerular cGMP levels in a concentration-dependent manner and with similar potencies (EC50 approximately 15-30 nM). The L-arginine analogue, N omega-nitro-L-arginine (L-NNA), reduced basal levels of cGMP and also totally inhibited ET-induced increases in cGMP as did methylene blue, an inhibitor of soluble guanylate cyclase. The effect of L-NNA was attenuated by L-arginine but not by D-arginine. The stimulation of cGMP accumulation by ET-3 was dependent on extracellular Ca2+ and was additive to atriopeptin III but not to acetylcholine. The ETA-selective antagonist, BQ 123, had no effect on ET-3-induced formation of cGMP. Glomerular membranes displayed high-affinity (Kd = 130-150 pM) and high-density (approximately 2.0 pmol/mg) binding sites for 125I-ET-1 and 125I-ET-3. ET-1, ET-3, and sarafotoxin S6c displaced 125I-ET-1 binding to glomerular membranes with similar affinities. BQ 123 had no effect on 125I-ET-1 binding. We conclude that ET increases cGMP levels in glomeruli by stimulating the formation of a nitric oxide-like factor that activates soluble guanylate cyclase. This effect of ET appears to be mediated by activation of ETB receptors and may serve to modulate the contractile effects of ET.

Frog glomerular vasotocin receptors resemble mammalian V1b receptors

1994 ◽  
Vol 267 (5) ◽  
pp. R1198-R1208 ◽  
Author(s):  
A. Ammar ◽  
R. M. Rajerison ◽  
S. Roseau ◽  
M. Bloch-Faure ◽  
D. Butlen
Keyword(s):  
Binding Sites ◽  
Inositol Phosphate ◽  
Rank Order ◽  
Enzyme Activation ◽  
Arginine Vasotocin ◽  
Pharmacological Properties ◽  
Nephron Segments ◽  
Inositol Phosphate Production ◽  
Vasopressin Receptors ◽  
Stimulation Of

Vasotocin receptors were investigated in glomeruli and nephron segments microdissected from collagenase-treated kidneys of Rana ridibunda, using [d(CH2)5Tyr(Me)2,Thr4,Orn8,125I-Tyr-NH2(9)]vasotocin (125I-OVTA) as a radioligand. Specific 125I-OVTA binding sites were found only in glomeruli and not in all tubule segments tested. Glomerular receptors exhibited the following stereospecificity for recognition of vasotocin analogues: Tyr-NH2(9)-LA-V1a > 125I-OVTA > arginine vasotocin (AVT) > or = [d(CH2)5Tyr-(Me)2]AVP > OVTA > or = [Phe2,Orn8]VT > oxytocin (OT) > or = [d(CH2)5-Sar7]AVP > desGly9[d(CH2)5Tyr(Et)2]VAVP > or = [d(CH2)5Tyr(Et)2]VAVP > AVP > [1-desamino-8-D-arginine]vasopressin (DDAVP) > [Thr4,Gly7]OT. In addition, vasotocin enhanced [3H]inositol phosphate production in sieved glomeruli labeled with myo-[3H]inositol; the rank order of structural vasotocin analogues for stimulation of phosphoinositidase C was [Phe2,Orn8]VT > AVT > OT > AVP > DDAVP, whereas [Thr4,Gly7]OT was almost inactive, and the rank order of antagonists for inhibition of hormone-induced enzyme activation was Tyr-NH2(9)-LA-V1a > [d(CH2)5Tyr(Me)2]AVP = OVTA > [d(CH2)5Sar7]AVP > [d(CH2)5Tyr(Et)2]VAVP > or = desGly9[d(CH2)5Tyr(Et)2]VAVP. Results indicate that the 125I-OVTA-labeled binding sites detected in frog glomeruli reveal the pharmacological properties of mammalian V1b-pituitary vasopressin receptors and might be physiological vasotocin receptors involved in phosphoinositidase C stimulation.

scholarly journals Binding of Adenosine Diphosphate (ADP) to Isolated Human Platelet Membranes

1977 ◽  
Author(s):  
J. Lips ◽  
J. J. Sixma
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Membrane Vesicles ◽  
Adenosine Diphosphate ◽  
Ph Optimum ◽  
High Affinity ◽  
Absolute Requirement ◽  
Millipore Filtration

Human platelet plasma membranes were isolated according to the glycerol loading technique of Barber and Jamieson. The binding of 14C ADP was studied with Millipore filtration in a Ca2+ and Mg2+ containing buffer at pH 7,4. At least two types of binding sites were found: A high affinity system with a maximum binding of 160 pMoles/mg protein and an association constant of 1,1 χ 106 M-1; and alow affinity system with a maximum binding of about 4500 pMoles/mg protein and an association constant of 0,6 χ 1θ4 M-1. The binding according to the high affinity system showed little temperature dependency (Q10 = 1,10). The pH optimum was at 7,3. Ca2+ ions were an absolute requirement for binding.Nucleoside diphosphokinase (NDPK) was found in the membrane vesicles. Evidence that this enzyme was not responsible for ADP binding was obtained. The enzyme is Mg2+ dependent and is inhibited by AMP, in contrast to ADP binding. The Q10 was 1,44.ADP binding was inhibited by ATP, IDP and β/γ-imidoadenosine triphosphate.

THYROID-STIMULATING ANTIBODIES IN GRAVES'S DISEASE AND THE EFFECT OF THYROTROPHIN-BINDING GLOBULINS ON THEIR DETERMINATION

1982 ◽  
Vol 92 (2) ◽  
pp. 175-184 ◽  
Author(s):  
J. BÍRÓ
Keyword(s):  
Binding Sites ◽  
High Capacity ◽  
Adenyl Cyclase ◽  
Point Of View ◽  
Human Thyroid ◽  
Binding Characteristics ◽  
High Affinity ◽  
Dose Dependent ◽  
Control Samples ◽  
Stimulation Of

Globulin preparations from the sera of 104 untreated patients with Graves's disease have been tested for their thyroid-stimulating antibody (TSAb) activities. Eighty-one of the samples (78%) were positive in the assay for the thyrotrophin-binding inhibitory immunoglobulins, 48 samples (46%) contained human thyroid adenyl cyclase stimulators (HTACS) and 71 (68%) contained human thyroid stimulators (HTS) measured as stimulation of colloid droplet formation in human thyroid slices. All 104 samples were positive in one or other of the assays, 29 (28%) were positive in all three assays and 38 (37%) in two. All samples were tested for their specific TSH-binding characteristics, 40 (38%) possessed 'B-type' binding sites (previously characterized as TSH-binding sites with low affinity but high capacity for the ligand) but the remaining 64 samples (62%) were no different from normal control samples and had 'A-type' binding sites (high affinity but low capacity binding sites for TSH). Samples without detectable thyrotrophin-binding inhibitory immunoglobulin did not contain B-type TSH-binding globulins. Globulins exhibiting B-type binding were more active in the HTACS and HTS assays. The B-type TSH-binding globulins have a characteristic, dose-dependent reducing effect on the human thyroid adenyl cyclase stimulation by TSH whereas A-type globulins do not. Globulins exhibiting B-type TSH-binding may therefore have a significant effect on assays for TSAb activities. The methods used to measure TSAb have been reviewed from this point of view.

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