Characteristics and developmental changes of corticotrophin-releasing hormone-binding sites in the fetal sheep anterior pituitary gland

1991 ◽  
Vol 130 (2) ◽  
pp. 223-229 ◽  
Author(s):  
F. Lü ◽  
K. Yang ◽  
J. R. G. Challis
Keyword(s):  
Binding Sites ◽  
Anterior Pituitary ◽  
Time Course ◽  
Scatchard Analysis ◽  
Maximum Output ◽  
Similar Time ◽  
Single Class ◽  
Fetal Sheep ◽  
Releasing Hormone ◽  
Receptor Number

ABSTRACT The responses of the fetal sheep pituitary to corticotrophin-releasing hormone (CRH) change during gestation with maximum output of ACTH around days 120–130, and decreased ACTH output near term. However, there is no information available concerning the extent to which these responses may be modulated by alterations in the number of CRH receptors. Therefore we measured specific CRH-binding sites, and changes in binding characteristics in membrane preparations from fetal sheep anterior pituitaries collected at days 65–70, 85–88, 100–110, 125–130 and at term (approximately 145 days). Binding assays were carried out using 125I-labelled Tyr-ovine CRH (125I-Tyr-oCRH), incubated with crude membrane fractions for 90 min at 22 °C. Binding was time- and temperature-dependent, linear with protein concentration, saturable and specific for oCRH. Scatchard analysis of binding data for individual tissues revealed a single class of CRH-binding sites with high affinity (Kd ≃1 nmol/l) that did not change significantly with gestational age. However, the number of CRH-binding sites increased progressively from days 65–70 to a maximum at days 125–130, then decreased at term. These results demonstrate the presence of specific CRH-binding sites in the fetal sheep anterior pituitary. Furthermore, the change in CRH receptor number with advancing pregnancy follows a similar time-course to the changes reported previously in responsiveness of the fetal sheep anterior pituitary to exogenous CRH stimulation in vivo. These results suggest that alterations in CRH receptor number may contribute to changes in responsiveness of the fetal sheep anterior pituitary to CRH during gestation. Journal of Endocrinology (1991) 130, 223–229

Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio)

1992 ◽  
Vol 70 (2) ◽  
pp. 268-274 ◽  
Author(s):  
Debananda Pati ◽  
Hamid R. Habibi
Keyword(s):  
Common Carp ◽  
Cyprinus Carpio ◽  
Binding Sites ◽  
Tissue Concentration ◽  
Gonadotropin Releasing Hormone ◽  
Scatchard Analysis ◽  
Single Class ◽  
Releasing Hormone ◽  
Carp Cyprinus Carpio

Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 °C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0–40.0 μg membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10−6 M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-Trp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites. sGnRH-A and cGnRH-II were found to bind with greater affinities than sGnRH and GnRH-ANT to the carp ovarian binding sites. These results provide for the first time characterization of GnRH binding sites in the ovary of a teleost species, Cyprinus carpio.Key words: gonadotropin-releasing hormone, luteinizing hormone releasing hormone, receptor, ovary, carp, Cyprinus carpio.

Characterization of gonadotropin-releasing hormone receptors in goldfish ovary: variation during follicular development

1993 ◽  
Vol 264 (2) ◽  
pp. R227-R234 ◽  
Author(s):  
D. Pati ◽  
H. R. Habibi
Keyword(s):  
Binding Sites ◽  
Hormone Receptors ◽  
Tissue Concentration ◽  
High Capacity ◽  
Follicular Development ◽  
Gonadotropin Releasing Hormone ◽  
Scatchard Analysis ◽  
Single Class ◽  
Releasing Hormone ◽  
High Affinity

Gonadotropin-releasing hormone (GnRH) receptors were characterized in the goldfish ovary using an analogue of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]GnRH; sGn-RH-A) as a labeled ligand. Binding of sGnRH-A to goldfish follicular membrane preparation was found to be saturable, reversible, and dependent on time, temperature, tissue concentration, and pH. Addition of unlabeled sGnRH-A displaced the bound 125I-labeled sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of two classes of binding sites, a high-affinity/low-capacity site and a low-affinity/high-capacity site, in the fully mature and less mature goldfish ovary. However, immature goldfish ovary contained only a single class of low-affinity binding sites. The equilibrium association constants (Ka) of the low-affinity sites were found to be similar in all follicular groups. However, there was a tendency for a higher Ka value of the high-affinity sites in the less mature follicles (0.5-0.95 mm) compared with the fully mature follicles (1.11-1.48 mm), although the differences were not statistically significant. Bound 125I-sGnRH-A was also found to be displaceable by sGnRH ([Trp7,Leu8]GnRH) and chicken GnRH-II (cGnRH-II; [His5,Trp7,Tyr8]GnRH), which occur naturally in the goldfish brain. sGnRH-A and cGnRH-II were found to bind with greater affinity than sGnRH to the goldfish ovarian GnRH binding sites.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

Ontogeny of the insulin response to glucose in fetal and adult sheep

1989 ◽  
Vol 67 (10) ◽  
pp. 1288-1293 ◽  
Author(s):  
Pamela E. Houghton ◽  
Thomas J. McDonald ◽  
John R. G. Challis
Keyword(s):  
Time Course ◽  
Bolus Injection ◽  
Peak Plasma ◽  
Femoral Vein ◽  
Insulin Response ◽  
Similar Time ◽  
Fetal Sheep ◽  
Adult Sheep ◽  
Glucose Ratio ◽  
Insulin Response To Glucose

The purpose of the present experiments was to examine in sheep whether the fetal insulin response to glucose was present by day 110 (d110) of pregnancy and whether the magnitude of the fetal insulin response changed between d110 and d145 (term). We also compared the responses observed in fetuses to those of adult nonpregnant sheep. Basal concentrations of glucose measured in plasma collected from the fetal femoral artery rose progressively between d110 and d145 of gestation, but did not attain the plasma glucose concentrations measured in adult sheep. Peak glucose concentrations in fetuses were achieved 10 min following the bolus injection of glucose (0.8 g/kg estimated fetal body weight) into the fetal femoral vein, and peak values increased with gestational age. Significantly higher peak glucose concentrations were achieved in adult sheep. The concentration of insulin rose rapidly in fetuses at d110, and a similar time course of insulin release in plasma was seen at all gestational ages. The peak plasma insulin concentrations were achieved at 20 min and were significantly greater in older (d140–145) than younger (d125–130) fetuses (p < 0.05). Peak insulin values in fetuses were much less than in adult sheep. In adult sheep glucose and insulin concentrations remained elevated at 120 min following the injection of glucose, whereas in the fetus the concentration of insulin had returned to preinjection values by 60 min. The insulin/glucose ratio did not change in fetal lambs over the last one third of gestation and was not different from the adult sheep. We conclude that (1) the fetal insulin response to an acute glucose load is present by d110 of gestation, and (2) the ratio of insulin released per unit glucose elevation did not change in fetal sheep over the last one third of gestation, nor between fetal and adult sheep.Key words: glucose, insulin, fetal sheep.

Identification and characterization of a corticotrophin-releasing hormone receptor in human placenta

1995 ◽  
Vol 133 (5) ◽  
pp. 591-597 ◽  
Author(s):  
Vicki L Clifton ◽  
Phillip C Owens ◽  
Phillip J Robinson ◽  
Roger Smith
Keyword(s):  
Human Placenta ◽  
Binding Sites ◽  
Hormone Receptor ◽  
Human Myometrium ◽  
Scatchard Analysis ◽  
Releasing Hormone ◽  
Rat Pituitary ◽  
Identification And Characterization ◽  
Crh Receptors

Clifton VL, Owens PC, Robinson PJ, Smith R. Identification and characterization of a corticotrophinreleasing hormone receptor in human placenta. Eur J Endocrinol 1995;133:591–7. ISSN 0804–4643 Corticotrophin-releasing hormone (CRH) causes vasodilatation in the human fetal–placental circulation and has paracrine actions in placental tissue, suggesting that CRH receptors may be present in the human placenta. We have now identified and characterized placental CRH binding sites and compared them to those described previously in human myometrium and rat pituitary. Radiolabelled ovine CRH binding to placental membranes was pH-, time-, temperature- and divalent cation-dependent and was reversible in the presence of 1 μmol/l unlabelled ovine CRH. Scatchard analysis of placentae delivered vaginally or by elective caesarean section revealed dissociation constants (Kd) of 214.5 ± 84 pmol/l (N = 8) and 45.4 ± 23.9 pmol/l (N = 9), respectively. The Kd for caesarean placental binding sites was similar to that of human myometrium (59.6 pmol/l, N = 3) and rat pituitary (82.5 pmol/l, N = 3) receptors. However, in vaginally delivered placentae the CRH binding sites had a much lower affinity (p < 0.05). The receptor densities (Bmax) of vaginally delivered and caesarean-delivered placentae were 28.6 ± 9.6 and 6.1 ± 2.8 fmol/mg, respectively (p < 0.05). Chemical cross-linking studies using disuccinimidyl suberate indicated that the molecular weight of the CRH receptor in the placenta and rat pituitary is 75 kD. We conclude that there is a high-affinity population of CRH binding sites in the human placenta that are physicochemically similar to pituitary and myometrial CRH receptors. The CRH receptor properties in the placenta change in response to labour, when CRH levels in maternal blood are highest, suggesting that placental CRH may regulate its receptor. R Smith, Endocrinology Unit, John Hunter Hospital, Locked Bag 1, Hunter Regional Mail Centre, Newcastle, NSW 2310, Australia

scholarly journals Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T Kitamura ◽  
A Tojo ◽  
T Kuwaki ◽  
S Chiba ◽  
K Miyazono ◽  
...  
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Scatchard Analysis ◽  
Hematopoietic Growth Factors ◽  
Single Class ◽  
Gm Csf ◽  
Molecular Weights ◽  
Human Erythropoietin ◽  
Macrophage Colony

Abstract We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

CORTICOSTERONE BINDING BY MOUSE SERA DURING FOETAL AND POST-NATAL DEVELOPMENT

1977 ◽  
Vol 84 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Lia Savu ◽  
Emmanuel Nunez ◽  
Max-Fernand Jayle
Keyword(s):  
Amniotic Fluid ◽  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Normal Adult ◽  
Scatchard Analysis ◽  
Mammalian Development ◽  
Single Class ◽  
Mean Values

ABSTRACT The binding properties of corticosterone binding globulin (CBG) of mouse sera have been studied by equilibrium dialysis and electrophoretic techniques, at different stages of foetal and post-natal development. Scatchard analysis has demonstrated in all cases a single class of high affinity saturable binding sites for corticosterone. Remarkable increases of the binding capacities were observed in the foetal and pregnant sera, as compared to normal adult and immature levels. The mean values of n1M1 × g−1 of serum proteins (concentration of binding sites, n1 × moles of binding proteins M1) were 21 10−8 in 14–19 day pregnant females, 17 10−8 in the amniotic fluid, 4.2 10−8 in 14–19 day embryos, and only 0.8 10−8 in the normal adult female. Neonatal mice, aged 0–6 days exhibited no CBG activities. The association constants showed values of 2.5–4.1 108 m−1 when measured with foetal sera, and of 1.2–2.1 108 m−1 with pregnant or control adult sera and with the amniotic fluid, at 25°C. Comparative electrophoretic, thermal denaturation and competition studies with foetal and pregnant plasma CBG's are also reported. The results are discussed in relation to the origin of CBG in the foetal serum, and also with respect to similar studies in the rat, guinea pig and man. The possible biological implications of serum steroid binding proteins in mammalian development are briefly outlined.

Involvement of estrogen receptors α and β in the regulation of cervical permeability

2000 ◽  
Vol 278 (4) ◽  
pp. C689-C696 ◽  
Author(s):  
George I. Gorodeski ◽  
Dipika Pal
Keyword(s):  
Estrogen Receptor ◽  
Binding Sites ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Scatchard Analysis ◽  
Single Class ◽  
Estrogen Receptor Modulators ◽  
Transcription Inhibitor ◽  
Estrogen Binding ◽  
In Cells

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888–C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of ∼0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time ≈ 4 h, and EC50≈ 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor α (αER) and estrogen receptor β (βER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of αER and βER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased βER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in αER or βER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an αER-dependent increase in transcription.

Relationship between muscarinic receptor occupancy and response in rat parotid acinar cells

1993 ◽  
Vol 265 (6) ◽  
pp. G1122-G1127 ◽  
Author(s):  
Y. Dai ◽  
B. J. Baum
Keyword(s):  
Binding Sites ◽  
Equilibrium Constants ◽  
Acinar Cells ◽  
Scatchard Analysis ◽  
Parotid Glands ◽  
Response Curves ◽  
Parotid Acinar Cells ◽  
Receptor Number ◽  
Rat Parotid ◽  
Q Values

To determine whether spare muscarinic cholinergic receptors (mAChRs) exist in rat parotid acinar cells, we examined the effect of propylbenzilylcholine mustard (PBCM) on agonist (carbachol)-stimulated inositol trisphosphate (IP3) formation and on mAChR number, using l-[N-methyl-3H]scopolamine methyl chloride (NMS)-binding assays. Treatment with PBCM (1, 3, 10, 30, 50 nM) for 15 min caused a 5, 22, 60, 66, and 72% decrease, respectively, in maximal IP3 formation stimulated by carbachol as well as a large reduction in the potency of carbachol in eliciting this response. Using these data, equilibrium constants (Ka) for activation of the mAChRs by carbachol were calculated. These Ka values agreed well with Kd values of high-affinity mAChR binding sites determined from carbachol displacement of [3H]NMS binding in parotid acinar cells. Reduction in mAChR number after PBCM treatment was determined by Scatchard analysis of specific [3H]-NMS binding sites and compared with the expected reduction (q values) calculated from dose-response curves for carbachol-stimulated IP3 formation before and after PBCM treatment. PBCM (1, 3, 10, 30 nM) decreased mAChR maximal binding in cells 47.5, 68.9, 82.4, and 85.3%, respectively, which did agree with the approximately 38, 70, 90, and 92% decrease in receptor number expected from the calculated q values. Data demonstrate that PBCM irreversibly inactivates mAChRs in rat parotid cells, and the decrease in receptor number, measured directly from [3H]NMS binding or calculated from receptor theory, is greater than that observed for stimulated IP3 production. These results suggest that a modest (30-40%) population of spare receptors exists for mAChR-mediated IP3 production in rat parotid glands.

Sign in / Sign up

Export Citation Format

Share Document