PHYSIOLOGICAL VARIATION IN ABUNDANCE OF OESTROGEN SPECIFIC HIGH-AFFINITY BINDING SITES IN HYPOTHALAMUS, PITUITARY AND UTERUS OF THE RAT

SUMMARY High-affinity binding of [2,4,6,7-3H]oestradiol-17β has been studied in cytosols prepared from hypothalami, pituitaries and uteri of female rats killed at different stages of the oestrous cycle, and in cytosols prepared from the hypothalami and pituitaries of male rats. In all cases the equilibrium dissociation constant of reaction was of the order of 10−10 mol/l. The number of available high affinity sites per tissue (n) varied with physiological state. In females, n fluctuated with the oestrous cycle. In hypothalamus and pituitary, n fell by about 60 and 40% respectively in pro-oestrus, replenishment occurred during oestrus but could be delayed by phenobarbitone administration during the afternoon of pro-oestrus. In the uterus, n varied biphasically, there being peaks during dioestrus and oestrus, and troughs at pro-oestrus and metoestrus. The numbers of available sites at metoestrus were 12·5 × 109, 10·6 × 1010 and 24·4 × 1010 for hypothalamus, pituitary and uterus respectively. In male rats, values for n were similar to those obtained for females at pro-oestrus (n/hypothalamus = 6·8 × 109, n/pituitary = 4·2 × 1010). Binding was oestrogen specific in all the tissues studied.

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Oxytocin receptors in the oviduct during the oestrous cycle of the ewe

Journal of Endocrinology ◽

10.1677/joe.0.1240353 ◽

1990 ◽

Vol 124 (3) ◽

pp. 353-359 ◽

Cited By ~ 7

Author(s):

V. J. Ayad ◽

S. A. McGoff ◽

D. C. Wathes

Keyword(s):

Binding Sites ◽

Luteal Phase ◽

Dissociation Constants ◽

Relative Concentration ◽

Oestrous Cycle ◽

Affinity Binding ◽

High Affinity Binding ◽

Lower Affinity ◽

High Affinity ◽

Oxytocin Receptors

ABSTRACT The presence of oxytocin receptors in ovine oviduct has been investigated. High-affinity binding sites for [3H]oxytocin were detected in crude membrane fractions prepared from the oviducts of ewes killed during the oestrous period. The dissociation constant calculated for these sites in competition studies was 1·7 nmol/l. Similar dissociation constants were calculated for [Arg8]-vasopressin and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4, Gly7]-oxytocin, indicating that these sites represent oxytocin receptors. At least one additional site of lower affinity and undetermined identity was present. The relative concentration of oxytocin-binding sites in preparations of oviduct membranes were estimated in ewes killed at different stages of the oestrous cycle using a single concentration of [3H]oxytocin. Binding was low during the luteal phase of the cycle but increased to a maximum at oestrus (77·7 fmol/mg protein). Binding fell after ovulation, reaching what appeared to be basal concentrations by the early luteal stage of the cycle. Binding to oviductal membranes from prepubertal, anoestrous and pregnant ewes was also low, but in anoestrous animals which had been treated with progesterone and oestrogen it was similar to values measured in ewes at oestrus. These results are consistent with the existence of oviductal oxytocin receptors which are regulated by ovarian steroids. We conclude that oxytocin receptors are present in the oviduct of the ewe around the time of ovulation. The significance of oxytocin to events taking place in the oviduct at this time remains to be determined. Journal of Endocrinology (1990) 124, 353–359

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High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648890 ◽

1994 ◽

Vol 72 (03) ◽

pp. 465-474 ◽

Cited By ~ 20

Author(s):

Neelesh Bangalore ◽

William N Drohan ◽

Carolyn L Orthner

Keyword(s):

Binding Sites ◽

Protein C ◽

Umbilical Vein ◽

Activated Protein C ◽

Affinity Binding ◽

Cell Binding ◽

High Affinity Binding ◽

High Affinity ◽

Gla Domain ◽

Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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