Characterization and localization of oxytocin receptors in the uterus and oviduct of the non-pregnant ewe using an iodinated receptor antagonist

1991 ◽  
Vol 128 (2) ◽  
pp. 187-NP ◽  
Author(s):  
V. J. Ayad ◽  
S. E. F. Guldenaar ◽  
D. C. Wathes
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Oxytocin Receptor ◽  
Binding Characteristics ◽  
High Affinity ◽  
Oxytocin Receptors ◽  
Secretory Glands ◽  
Pregnant Ewe ◽  
Autoradiographic Technique ◽  
Specific Labelling

ABSTRACT Some of the binding characteristics of a novel oxytocin receptor ligand 125I-labelled [1-(β-mercapto-β, β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2,Thr4,Orn8,Tyr9-NH2]-vasotocin ([125I]OTA) have been determined in the sheep uterus. The compound was subsequently used for the autoradiographic localization of oxytocin receptors in the uterus and oviduct of the ewe. Specific binding of [125I]OTA to crude membrane fractions of ovine endometrium was time-dependent and was unaffected by the addition of cations to incubation media. Endometrial membranes contained a single population of saturable, high-affinity binding sites for the iodinated ligand (dissociation constant (Kd) 0·23±0·08 nmol/l) and unlabelled oxytocin competed with [125I]OTA for binding sites with high affinity (Kd 1·29±0·4 nmol/l) in the presence of Mg2+ In contrast, unlabelled OTA was able to compete with high affinity (Kd 1·13±0·16 nmol/l) in the absence of cation. Competition studies with a number of oxytocin analogues and related peptides and the tissue distribution of [125I]OTA binding sites also indicated that [125I]OTA bound to the ovine oxytocin receptor. This was further validated by autoradiographic studies which showed specific labelling with [125I]OTA to be greater to uterus and oviduct obtained from ewes which had been killed within 2 days of oestrus than to similar tissue from ewes killed during the luteal phase. In both the ampullary and isthmic regions of the oviduct and the myometrium, [125I]OTA binding sites were confined to smooth muscle. Endometrial binding sites for [125I]OTA were consistently located on the luminal epithelium and epithelial cells lining secretory glands. In addition, in one ewe which had been killed 2 days after cloprostenol treatment, stromal cells were labelled in a caruncular region of the endometrium. The consistency of this observation between similar animals remains to be determined. The autoradiographic technique demonstrated appears sufficiently sensitive to allow further studies into the distribution of the endometrial oxytocin receptor throughout the oestrous cycle, and into its regulation at luteolysis and during the establishment of pregnancy. Journal of Endocrinology (1991) 128, 187–195

Characterization and localization of a putative oxytocin receptor in the cervix of the oestrous ewe

1994 ◽  
Vol 142 (3) ◽  
pp. 397-405 ◽  
Author(s):  
E L Matthews ◽  
V J Ayad
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Luteal Phase ◽  
Specific Binding ◽  
Oxytocin Receptor ◽  
Oestrous Cycle ◽  
Secretory Cells ◽  
Cervical Tissue ◽  
High Affinity ◽  
General Decline

Abstract The purpose of the present study was to investigate the presence of high-affinity oxytocin-binding sites (putative oxytocin receptors) in the cervix of the non-pregnant ewe. [3H]Oxytocin binding to the peripheral layers of cervical tissue (comprising the serosal layer and the least dense collagenous and muscular layers) and the remaining dense collagenous cervical tissue were studied separately. [3H] Oxytocin-binding sites were detected in membrane fractions prepared from both of these regions, but binding to the peripheral cervix was variable and binding site concentrations were low, hence these were not characterized further. A high-affinity oxytocin-binding site, having a dissociation constant of 1·8 nmol/l, was characterized in the dense collagenous regions of the cervix of ewes killed during the oestrous period. Similar dissociation constants were determined for [Arg8]-vasopressin and the oxytocin-specific agonist [Thr4, Gly7]-oxytocin in competition studies. [3H] Oxytocin binding to peripheral cervical tissue and to the dense collagenous cervix was generally low or undetectable during the luteal phase, but increased in both tissues around the time of luteolysis. Although specific binding to both tissues was variable during the oestrous period, it was higher at this time than during the luteal phase. [3H] Oxytocin-binding site concentrations were also found to be higher within the inner dense collagenous cervix of oestrous ewes than of pregnant, ovariectomized or anoestrous animals. During the oestrous cycle, oxytocin-binding site concentrations reached a maximum in the dense collagenous cervical tissue on the day of oestrus (141·8 ±44 (s.e.m.) fmol/mg protein), showing a general decline during the following days back to luteal phase values. This compared with concentrations of 513·3 ±132·1 and 216·1 ± 13·9 fmol/mg protein, measured for comparative purposes in endometrial and myometrial membrane preparations, respectively, on the day of oestrus in the same group of ewes. However, in membrane preparations of peripheral cervical tissue higher concentrations were measured on day 14 than on the following 2 days and maximal concentrations were not reached until the day after oestrus (52·7 ± 4·2 fmol/mg protein). Concentrations were maintained at similar values during the subsequent 2 days and significant specific binding was still measurable in both regions of the cervix on day 5. The localization of oxytocin-binding sites within dense collagenous cervical tissue was investigated autoradiographically using the 125I-labelled specific oxytocin receptor antagonist [1(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2, Thr4, Orn8, Tyr9 -NH2]-vasotocin. The only clear specific labelling was localized to the luminal epithelium of the uterine section of the cervix of oestrous ewes, with labelling in ewes in the luteal phase clearly reduced or absent. The results demonstrate the presence of a high-affinity oxytocin-binding site within the cervix of the oestrous ewe which is associated with secretory cells and which undergoes similar changes in concentration during the oestrous cycle to uterine oxytocin receptor sites. The significance of this novel putative site of oxytocin action remains to be established. Journal of Endocrinology (1994) 142, 397–405

Human chorionic gonadotropin binding sites in the human endometrium

1993 ◽  
Vol 129 (1) ◽  
pp. 15-19 ◽  
Author(s):  
S Bhattacharya ◽  
J Banerjee ◽  
S Sen ◽  
PR Manna
Keyword(s):  
Metal Ions ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Endometrium ◽  
Binding Characteristics ◽  
High Affinity ◽  
Specific Binding Sites

The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. We have identified hCG binding sites in the human endometrium collected from 35–42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5 × 10−10 mol/l and in anovulatory women to be 3.1 × 10−10 mol/l. The maximum binding capacity varied considerably between ovulatory (3.85 nmol/kg protein) and anovulatory (6.12 nmol/kg protein) endometrium. Among the divalent metal ions tested (Zn2+, Mg2+, Mn2+, Ca2+—4 mol/l), Zn2+ effected a remarkable increase in [125I]hCG binding to the endometrium (p<0.005) whereas Mn2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [125I]hCG binding to endometrium.

scholarly journals Two Binding Sites for [3H]PBR28 in Human Brain: Implications for TSPO PET Imaging of Neuroinflammation

2010 ◽  
Vol 30 (9) ◽  
pp. 1608-1618 ◽  
Author(s):  
David R Owen ◽  
Owain W Howell ◽  
Sac-Pham Tang ◽  
Lisa A Wells ◽  
Idriss Bennacef ◽  
...  
Keyword(s):  
Binding Sites ◽  
Marked Reduction ◽  
Specific Binding ◽  
Translocator Protein ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Binding Characteristics ◽  
High Affinity ◽  
High Affinity Site ◽  
Site Binding

[11C]PBR28, a radioligand targeting the translocator protein (TSPO), does not produce a specific binding signal in approximately 14% of healthy volunteers. This phenomenon has not been reported for [11C]PK11195, another TSPO radioligand. We measured the specific binding signals with [3H]PK11195 and [3H]PBR28 in brain tissue from 22 donors. Overall, 23% of the samples did not generate a visually detectable specific autoradiographic signal with [3H]PBR28, although all samples showed [3H]PK11195 binding. There was a marked reduction in the affinity of [3H]PBR28 for TSPO in samples with no visible [3H]PBR28 autoradiographic signal ( K i=188±15.6 nmol/L), relative to those showing normal signal ( K i=3.4±0.5 nmol/L, P<0.001). Of this latter group, [3H]PBR28 bound with a two-site fit in 40% of cases, with affinities ( K i) of 4.0±2.4 nmol/L (high-affinity site) and 313±77 nmol/L (low-affinity site). There was no difference in Kd or Bmax for [3H]PK11195 in samples showing no [3H]PBR28 autoradiographic signal relative to those showing normal [3H]PBR28 autoradiographic signal. [3H]PK11195 bound with a single site for all samples. The existence of three different binding patterns with PBR28 (high-affinity binding (46%), low-affinity binding (23%), and two-site binding (31%)) suggests that a reduction in [11C]PBR28 binding may not be interpreted simply as a reduction in TSPO density. The functional significance of differences in binding characteristics warrants further investigation.

Characterization of endometrial and myometrial oxytocin receptors in the non-pregnant ewe

1989 ◽  
Vol 123 (1) ◽  
pp. 11-18 ◽  
Author(s):  
V. J. Ayad ◽  
D. C. Wathes
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Divalent Cations ◽  
Single Site ◽  
High Affinity ◽  
Oxytocin Receptors ◽  
Pregnant Ewe

ABSTRACT Oxytocin-binding sites in the endometrium and myometrium of the non-pregnant ewe were characterized. [3H]Oxytocin bound to a single site in both tissues with high affinity; dissociation constants were determined to be 1·96 nmol/l in endometrium and 2·12 nmol/l in myometrium. Oxytocin binding was enhanced by divalent cations with a similar order of potency in both tissues: Co2+> Mn2+> Ni2+> Mg2+ > Zn2+> Ca2+. The endometrial and myometrial binding sites showed the same specificity for oxytocin analogues and related peptides, having high affinity for oxytocin, [Arg8]-vasopressin, [Lys8]-vasopressin, and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4,Gly7]-oxytocin. The results suggest that oxytocin receptors present in the endometrium and myometrium of the ewe are similar both to each other and to classical oxytocin receptors. Journal of Endocrinology (1989) 123, 11–18

Fasting alters somatostatin binding to liver membranes of rainbow trout (Oncorhynchus mykiss)

1996 ◽  
Vol 150 (2) ◽  
pp. 179-186 ◽  
Author(s):  
M J Pesek ◽  
M A Sheridan
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
High Affinity ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
C Terminus ◽  
Liver Membranes

Abstract Somatostatins are a diverse family of peptides that influence various aspects of animal growth, development, and metabolism. Recent work in our laboratory has shown that somatostatins stimulate hepatic lipolysis in rainbow trout. In this study we characterized somatostatin-binding sites in trout hepatic membrane preparations. We also examined changes in binding characteristics brought about by food deprivation. Binding of [Tyr11]-somatostatin-14 (SS-14) was saturable, reversible, and time- and temperature-dependent. Under optimal conditions, [Tyr11]-SS-14 specific binding averaged 5·7 ± 0·3%. While SS-14 and SS-28 (an N-terminally extended form of SS-14 and derived from the same gene as SS-14) displaced [Tyr11]-SS-14 specific binding (ED50 values of approximately 50 nm and 100 nm respectively), salmon SS-25 (containing [Tyr7,Gly10]-SS-14 at its C terminus and presumably derived from a gene different from that giving rise to SS-14/SS-28), except at pharmacological concentrations, did not. Significant specific binding was also detected in brain, esophagus, stomach, upper and lower intestine, pancreas, and adipose tissue. Scatchard analysis suggested the existence of two classes of hepatic somatostatin-binding sites: a high-affinity site with a Kd of 23 nm and Bmax of 1·4 pmol/mg protein and a low-affinity site with a Kd of 379 nm and Bmax of 4·9 pmol/mg protein. Fasting resulted in reduced growth and elevated plasma levels of SS-14 compared with fed animals. SS-14 binding capacity of the high-affinity class in liver membranes isolated from fasted fish increased by 120% over that from fed counter-parts. No difference in Kd for the high-affinity binding class or in either Kd or Bmax of the low-affinity class was noted between fasted and fed animals. These data support the role of the liver as a target of somatostatin and suggest that fasting enhances hepatic sensitivity to SS-14 binding. Journal of Endocrinology (1996) 150, 179–186

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

scholarly journals Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity

1984 ◽  
Vol 222 (3) ◽  
pp. 639-647 ◽  
Author(s):  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Sites ◽  
Maximal Activity ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Male Rats ◽  
Heart Mitochondria ◽  
Binding Characteristics ◽  
Functional Sites ◽  
High Affinity ◽  
Malonyl Coa

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5′-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

scholarly journals Binding of Calcium to Fibrinogen: Some Related Properties

1977 ◽  
Author(s):  
G. Marguerie
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Structural Features ◽  
Salt Bridges ◽  
High Affinity ◽  
Direct Titration ◽  
Specific Binding Sites ◽  
Calcium Binding Sites

The calcium binding properties of bovin fibrinogen have been studied using equilibrium dialysis method. At pH 7.5 fibrinogen has 3 specific calcium binding sites of high affinity and several non specific binding sites of low affinity. Direct titration of the calcium induced proton release indicates that the binding center is a chelate. Thermal an acid denaturation is found to be markedly influenced by the presence of Ca++, suggesting that structural features are related to the binding. However the circular dichroism spectra show that no generalized conformational change is induced when Ca++ is bound to the protein.The plasminic digestion of fibrinogen is also found to be specificaly influenced by Ca++. The velocity of the initial cleavages is slightly reduced in the presence of calcium. It is therefore suggested that the C-terminal part of the Aα chain is involved in the binding.Considering the dimeric structure of the fibrinogen molecule, the presence of only 3 calcium binding sites of high affinity suggests the existence of “salt bridges” between the constitutive polypeptide chains.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

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