An improved method for the study of high-affinity steroid binding: Oestradiol binding in brain and pituitary

Ginsburg, M.; Greenstein, Ben; MacLusky, Neil J.; Morris, Ian D.; Thomas, Patrick J.

doi:10.1016/0039-128x(74)90166-4

Cited by 34 publications

(44 citation statements)

References 17 publications

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“…Additional analysis using the computer program LIGAND (Munson and Rodbard 1980) In Table 2 Tolton et al 1985). This is similar to the relationship determined for rat hypothalamic and uterine tissue as reported by Ginsburg et al (1974) Affinity constants for the AP were found…”

supporting

confidence: 73%

“…This does not agree with results reported by Diekman and Anderson (1982a) (Glass et al 1984). In female rats, utilizing gel filtration methods to assay E2R, Ka values for samples from the pituitary were also reported to be greater than Ka values obtained from hypothalamic tissue (Ginsburg et al 1974 al. 1951) or an increase in crosslinking of structural proteins (Heathcote and Grant 1981 For personal use only.…”

mentioning

confidence: 99%

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Estrogen Receptor Content of Regions of the Hypothalamus and Pituitary of the Gilt From Four to Twenty-Eight Weeks of Age

Evans¹,

Christopherson²,

Aherne³

1989

Can. J. Anim. Sci.

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Estradiol-17β receptors (E2R) in 100 000 × g supernatant fractions from the hypothalamus and pituitary were determined for groups of five gilts, slaughtered at 4-wk intervals from 4 to 28 wk of age. Tissue samples were separated into the anterior hypothalamus (AH), the central hypothalamus (CH), posterior hypothalamus (PH), anterior pituitary (AP) and posterior pituitary (PP). After saturation binding assays were conducted, E2R concentrations were determined by Scatchard analysis. When expressed on a fmol mg−1 protein basis there was no increase in receptor concentration with age (P > 0.05); however, AP had higher concentrations of receptors than the AH, CH or PH (10.6 ± 1.4 vs. 6.5 ± 0.9, 6.7 ± 0.7 and 6.2 ± 0.6, P < 0.01). Posterior pituitary receptor levels remained low during maturation of the gilt with affinity constant (Ka) values similar to that obtained for the AP. Affinity constant values were found not to change with age; however, AP values were greater than that of the hypothalamus samples (55.2 ± 2.7 vs. 4.8 ± 0.2, × 108M−1). On a total tissue basis AH and CH had significant increases in receptor content from 4 to 28 wk (41.4 to 138.2 and 31.6 to 163.4 fmol, P < 0.05). It was also determined that the soluble protein content per gram wet tissue decreased with age in all regions of the hypothalamus and therefore may mask increases in receptor numbers. Keywords: Estradiol, receptor, gilt, hypothalamus, pituitary, swine

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supporting

confidence: 73%

mentioning

confidence: 99%

Estrogen Receptor Content of Regions of the Hypothalamus and Pituitary of the Gilt From Four to Twenty-Eight Weeks of Age

Evans¹,

Christopherson²,

Aherne³

1989

Can. J. Anim. Sci.

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“…The diluted samples were incubated for 4 h at 4°C with 0.4 nM 'H R5020 (87 Ci/mmol, New England Nuclear, or 57 Ci/mmol, gift from J.P. Raynaud) or 0.4 nM 'H R5020 along with 0.2 p M unla beled R5020 to measure nonspecific binding. The 3H R5020 bound to receptors was separated from the free ligand by Sephadex LH-20 chromatography according to the general procedure of Ginsburg et ®b [9]. A 200-pl aliquot of TK.48DG buffer was applied to a 0.5x7 cm column of Sephadex LH-20 (Pharmacia), equilibrated with TK.4G buffer.…”

Section: Progestin Exchange a Waymentioning

confidence: 99%

Nuclear Progestin Receptors in Rat Brain and Pituitary

et al. 1982

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Progestin receptors in rat brain and pituitary cell nuclei were measured with an in vitro exchange assay in order to examine the role of estrogen priming and of endogenous and exogenous progesterone in determining the amount of receptor which reaches the nuclear compartment. Progesterone administration causes progestin receptors, demonstrable by exchange, to appear in the cell nuclear fraction from rat pituitary, hypothalamus-preoptic area (HYP-POA), and cerebral cortex, all of which contain cytosol progestin receptor sites. The amount of progestin receptor translocated to nuclei by 1 mg of progesterone increases after estrogen priming in pituitary and HYP-POA, tissues which contain progestin receptors inducible by estradiol. The increase in nuclear progestin receptors after progesterone is proportionally the same in estrogen-primed and in unprimed HYP-POA as well as in the cerebral cortex, where noninducible progestin receptors are found. Further characterization of nuclear progestin receptors reveals that those translocated to cell nuclei are differentialy extracted by 0.4 M KCl and that high estrogen treatment in the absence of progesterone appears to give rise to some progestin receptor translocation to nuclei. The influence of both estrogen priming and progesterone release is evident in HYP-POA and pituitary cell nuclei assay by progestin exchange from rats killed at various stages of the estrous cycle. The amount of nuclear progestin receptor increases from diestrus to noon on proestrus, due apparently to endogenous estrogen priming, since progesterone levels remain the same and cytosol progestin receptors are known to increase in this time. Nuclear progestin receptors increase from noon to 16:30 h on proestrus in parallel with the proestrus rise of progesterone. This increase precedes the normal appearance of estrous behavior on the evening of proestrus. When estrogen-primed ovariectomized rats are given varying doses of progesterone and tested for sexual behavior or killed for progestin receptor exchange assays, nuclear progestin receptors are found to increase in the same way as the dose dependence of proceptive sexual behavior. Thus, the translocation of progestin receptors to MBH-POA cell nuclei may be an important step in the activation of proceptive sexual behavior by progesterone.

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“…Methods [2,4,6,73H1-oestradiol (85 Ci/mmol), obtained from the Radiochemical Centre, Amersham, England, was repurified and stored as described before (Ginsburg, Greenstein, MacLusky, Morris & Thomas, 1974 (Ginsburg et al, 1974). Homogenates were centrifuged (110,000 ravx g x 1 h) to prepare cytosols.…”

Section: Introductionmentioning

confidence: 99%

“…Homogenates were centrifuged (110,000 ravx g x 1 h) to prepare cytosols. [3H1-oestradiol: equilibrium dissociation constants of reaction were determined as described in detail elsewhere (Ginsburg et al, 1974). In summary, 0.2 ml aliquots of cytosols were incubated (300C) with 0.1 ml aliquots of [3H1-oestradiol (10-9 M to 3 x 10-10 M) in buffer for 10 min (hypothalamus), 20 min (pituitary) and 30 min (cortex and amygdala); times that are more than enough to allow equilibrium between the high affinity receptor and the hormone to be reached.…”

Section: Introductionmentioning

confidence: 99%