Ernest J. Peck

Heterogeneity of estrogen receptors in the cytosol and nuclear fractions of the rat uterus.

Multiple species of estrogen binding sites have been demonstrated in cytosol and nuclear fractions of uteri from immature and adult ovariectomized female rats. Equilibrium binding analyses of uterine cytosol yielded two binding sites, I and II, with dissociation constants of 0.8 and 33 nM respectively. The high affinity cytosol site (0.8 nM - Type I) translocated to the nuclear compartment following estrogen treatment in vivo and appears to represent the classical estrogen receptor which can be measured by 3H-estradiol exchange. Type II sites remain in the cytosol after estrogen injection. A third binding component was observed in the nuclear compartment (nuclear Type II) which binds 3H-estradiol at 4° and displayed cooperative binding characteristics. The presence of these sites in both cytosol and nuclear compartments complicates the accurate measurement and differentiation of these sites. Valid estimates of binding parameters for cytosol Type I and II sites may be obtained by saturation analyses over a wide range of 3H-estradiol concentrations (0.05–40 nM). Nuclear Type I can be differentiated from nuclear Type II by performing saturation analysis under exchange conditions which measure both Type I and II sites and comparing the values obtained when the assay is performed at 4°C which measures only Type II sites.

Regulation of estrogen receptor replenishment by progesterone.

The cyclic changes in female reproductive tissues have been studied extensively by reproductive biologists. The periodic variations in blood levels of estrogen and progesterone are believed to be responsible for the changes observed in uterine tissue throughout the estrous and menstrual cycles. With the discovery of steroid hormone receptors, it became apparent that variations in tissue levels of hormone receptors might also act as a regulatory step in the control of reproductive cyclicity. It has been suggested that progesterone might antagonize the action of estrogen by reducing the amount of cytoplasmic estrogen receptor, Rc.i-3 Recent work from our laboratory indicates that this is one of the mechanisms by which progesterone and nonsteroidal antiestrogens exert their antagonistic eff e c t ~ . ~ ~ Another possibility is that progesterone might interfere with the nuclear binding of the receptor-estrogen complex and thus reduce or modify its effects on nucleus-mediated events. In this paper, we have examined the influence of progesterone on estrogen receptor replenishment and nuclear binding in immature and adult rats.

Biphasic response of hippocampal pyramidal neurons to GABA

GABA released either iontophoretically or synaptically near pyramidal neurons in the CA1 region of the rat hippocampal slice could produce a biphasic response: a hyperpolarization followed by a depolarization. The depolarizing component elicited by either method was accompanied by an increased membrane conductance, and a reduction in neuronal discharge. The depolarization was reversed at a potential which was less negative than the resting membrane potential; it was blocked by antagonists of GABA action such as picrotoxin; it was sensitive to manipulation of extracellular chloride concentration; and it persisted in the presence of concentrations of cobalt or manganese which were sufficient to block evoked synaptic activity. Iontophoresis of GABA near the apical dendrites elicited an initial depolarization rather than an initial hyperpolarization, suggesting a dendritic origin for the depolarizing component. Together, these results suggest that GABA can produce, in the same neuron, both hyperpolarizing and depolarizing responses which depend at least in part upon changes in chloride conductances.

Receptor progesterone complex in the nuclear fraction of the rat uterus: Demonstration by 3H-progesterone exchange

We have previously shown that 3H-estradiol exchange can be used to measure the quantity of estrogen receptor complex in the nuclear fraction of target tissue cells. This method has been modified for the measurement of the progesterone receptor complex (Rn·P) in the nuclear fraction of uterine cells. Nuclear fractions were incubated for 5 hrs. at 15°C in the presence of varying concentrations of 3H-progesterone (3H-P) with or without a 250-fold excess of non-labeled progesterone (P). Rn·P was determined by subtracting the 3H-P bound in the presence of excess P (non-specific binding) from 3H-P bound in the absence of excess P (total binding). All Rn·P studies were done in adult castrate female rats that had received estradiol benzoate (0.4 mg) one week before use. The quantity of Rn·P increased in the uterine nuclear fractions by 280% 30 min. after injection of 5 mg of P. Rn·P was not increased in muscle or fat pad by this treatment. Injections of corticosterone (B), cortisol (F), dexamethasone (Dx) or testosterone (T) failed to increase Rn·P. The exchange reaction was specific for P; B, F, Dx or T did not compete. These results demonstrate the existence of a low capacity, high affinity, stereospecific progesterone binding site in the nuclear fraction of the uterus.

Estrogens and the Hypothalamus: Nuclear Receptor and RNA Polymerase Activation

AbstractTo further the understanding of estrogen action in the central nervous system, we have developed a procedure for quantitation of nuclear estrogen receptor (RnE) in adult rat brain. Crude chromatin is separated from soluble and membranous fractions by centrifugation of brain homogenate through 1.2 M sucrose pads. Incubation of the pellet with [3H] estradiol at elevated temperature and precipitation of receptor-steroid complexes with protamine sulfate allows measurement of RnE. The yield of DNA by this procedure is 70–80% and the amount of RnE measured is linear with respect to the amount of DNA added. Using this procedure, we have measured hypothalamic RnE in the ovariectomized adult rat as a function of time after injection of 5 ug estradiol and found a peak RnE accumulation of about 30 fmol/hypothalamus at 1 h. By 12 h, the levels of RnE return to control (uninjected) values.We have also measured the time course of estradiol induced activation of hypothalamic endogenous nuclear RNA polymerases I ...

Alterations in iodinated cell surface proteins during myogenesis.

Lactoperoxidase catalysed iodination was used to label surface proteins of chick embryo muscle cells during myogenesis. Both quantitative and qualitative changes were observed between 125I-labelled surface proteins of pre-fusion, mid-fusion, and post-fusion cells. Significantly, two bands at 245,000 molecular weight were present at pre-fusion but were observed as a single band at mid- and post-fusion. Radioactivity in this band increased selectively at post-fusion with a concomitant increase in lower molecular weight labelled proteins.

Differential Solubilization of γ‐Aminobutyric Acid Receptive Sites from Membranes of Mammalian Brain

Sodium‐dependent (+Na) and sodium‐independent (‐Na) receptive sites for γ‐aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized as to binding constants, pharmacologic specificities, and sodium dependence. The SPM fraction was then treated with various concentrations of Triton X‐100 resulting in the loss of pharmacologic specificity, binding characteristics, and sodium dependence associated with +Na GABA receptive sites in SPM. The resulting junctional complex preparation (JC), i.e., a fraction enriched in junctional complexes, possessed only the pharmacologic specificity and binding constants associated with Na receptive sites whether assayed in the presence or absence of 100 mwNaC1. This is probably due to the detergent dispersal or solubilization of the +Na GABA receptive site. The binding constants, KD and Bmax, for –Na GABA binding in SPM were 170 nM and 4.4 pmol/mg protein, while in JC they were 186 nM and 3.7 pmol/mg protein. Under repeated washing the KD was reduced to 60 ± 6.9 nM and the Bmax was reduced to 2.5 ± 0.5 pmol/mg protein in JC, probably owing to the removal of endogenous ligand or inhibitor, and not to inhibition by residual Triton X‐100. Multiple extraction with 0.1% or 0.5% Triton X‐100 did not alter the KB or Bmax, values for the binding of [3H] GABA to JC. Sodium‐independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.

Resolution of Estrogen Binding Species in Hypothalamus and Pituitary

In addition to the classic Type I estrogen receptor, a second estrogen binding species has been reported in rat uterine nuclear material. We have examined nuclear material from hypothalamus and pituitary for the presence of Type II estrogen receptor by the sucrose pad/exchange assay. Extensive (0.05-40.0 nM) saturation analyses were performed on crude chromatin isolated from hypothalami and pituitaries of hyperestrogenized ovariectomized rats. Analysis of data by an adaptation of the graphical method of Rosenthal suggests that there is only a single class of estrogen binding sites in these tissues with a Kd (0.2 nM) close to that reported for Type I receptors in other systems. However, a definitive resolution of the binding component(s) was not possible due to noise in the upper regions of the saturation plot. Therefore, we pooled 7 hypothalamic and 4 pituitary saturation experiments and analyzed the data with LIGAND, a nonlinear curve fitting program. Computer generated curves indicate that the data from both target tissues are approximated most closely by a model describing a single high affinity binding species (Type I) (Kd = 0.17 - 0.38 nM) and a linear or very low affinity nonspecific binding component. Thus, we conclude that there is no evidence for the presence of a second or lower affinity estrogen binding component in hypothalamus or pituitary. The absence of this binding species in these two estrogen target tissues is consistent with the concept that Type II sites may be involved in estrogens control of tissue hypertrophy and hyperplasia--estrogen responses not found in hypothalamus or pituitary.

Steroid Receptor Characterization and Measurement

A complete understanding of the relationship between steroid receptor binding and the mechanism of hormone action depends on valid characterization and accurate measurement of steroid receptors. In this chapter the criteria and methods by which this can be accomplished are discussed. These provide the necessary background for the studies that will be presented in subsequent chapters and represent an expression of previous work published on this topic (Clark and Peck, 1977d, 1978a).

Chapter 24 Methods for Assessing the Binding of Steroid Hormones in Nuclei and Chromatin

Publisher Summary This chapter describes the methods for assessing the binding of steroid hormones in nuclei and chromatin. Steroid hormone responsive cells contain macromolecules called receptors. These receptors represent the primary mechanism by which target cells detect and respond to hormones. An exchange assay consists of the preparation of subcellular fractions at low temperature, exchange at elevated temperature, and subsequent work-up and processing at low temperature. The chapter describes the steroid exchange methodology. The [ 3 H]estradiol exchange assay is useful for measuring nuclear receptor–estrogen complexes in the uterus, in the pituitary and hypothalamus, in lactating mammary gland, in the corpus luteum, in chick oviduct, and in Leydig cell tumors. The slow rate of dissociation at lower temperatures allows isolation and processing of subcellular fractions such as cytosols and nuclei without appreciable alternation of existing receptor–steroid complexes. The rapid rate of dissociation at elevated temperatures allows the exchange of added 3 H labeled steroid for unlabeled steroid occupying receptor sites.