Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Testicular androgen-binding protein (ABP): comparison of abp in rabbit testis and epididymis with a similar androgen-binding protein (TeBG) in rabbit serum

Testicular androgen-binding proteins (ABP) in rabbit testis, caput epididymis and efferent duct fluid (EDF) were compared to a similar androgen-binding protein TeBG) in rabbit serum. The affinity of these proteins for 5α-dihydrotestosterone (DHT) at 0 °C (K_a^ABP = 1.6 × 10⁹ M^?1 and K_a^TeBG = 1.9 × 10⁹ M^?1) and their steroid specificities were similar (DHT > androstanediol > progesterone and androstenedione). ABP and TeBG had also almost identical Stokes radii (42.8 ± 1.2 and 43.9 ± 0.8 Å, respectively), sedimentation coefficients (4.7 ± 0.2 S and 4.4 ± 0.2 S, respectively) and electrophoretic mobility (R_f = 0.4 in 6$\text{1}\text{2}$% polyacrylamide gels). Calculation of molecular weights from Stokes radii and sedimentation rates indicated a molecular weight of 74,000 (69,000–78,000) for TeBG and 76,000 (71,000–82,000) for ABP. The corresponding frictional ratios were 1.61 for TeBG and 1.55 for ABP assuming a partial specific volume (–v) of 0.70 cm³/g. Polyacrylamide gel electrophoresis (PAGE) at different gel concentrations gave a mean molecular radius of 2.74 nm, also indicating a molecular weight of about 75,000 (–v = 0.70 cm³/g). ABP and TeBG could not be separated by PAGE; however, partial separation of ABP and TeBG was achieved by isoelectric focusing and ion-exchange chromatography on DEAE-cellulose. TeBG focused at pH 5.4, whereas ABP formed a distinct peak of bound radioactivity at pH 4.7. Also by ion-exchange chromatography, ABP in both testis and epididymal supernatants was shown to have an apparently higher surface charge than TeBG in rabbit serum.The concentration of ABP in efferent duct fluid (2 × 10^?7 M = 60 pmol/mg protein) was much higher than TeBG in male rabbit serum (5.2 × 10^?8 M = 0.7 pmol/mg protein). These findings ruled against the possibility that ABP in the testis and epididymis could have been derived directly from serum. It is concluded that ABP and TeBG are very similar if not identical proteins both serving as transport and carrier proteins in their respective compartments.     

Further characterization of the androgen receptor in rat testis.

Immature rat testes contain a specific binding protein for testosterone (T) and 5alpha-dihyrotestosterone (DHT) with physico-chemical properties similar to the cytoplasmic androgen receptors in the epididymis and ventral prostate but different from the testicular androgen-binding protein (ABP). Like the androgen receptors in the prostate and epididymis, it has a sedimentation coefficient of about 7 S at low ionic strength, is eluted in or close to the void volume on Sephadex G-200 gel filtration (Stokes radius greater than 80 A), has an isoelectric point of about 5.6-6.0 (mean) 5.8 and a relative mobility (Rf) of 0.4 in 3.25% acrylamide gels. Following the injection of 3H-labeled testosterone, T and DHT are bound selectively by the receptor. Relatively more [3H]T than [3H]DHT is present in bound and free fractions as well as in total testicular 105,000 g supernatant. Similar results are obtained from testicular incubations with equimolar amounts of [3H]T and [3H]DHT at 0 degrees C in vitro. Saturation of receptor sites is achieved by incubation of testis supernatants with increasing amounts of [3H]T at 0 degrees C. The number of available binding sites following post-hypophysectomy regression is estimated to be about 9 fmoles/mg protein, and the apparent equilibrium constant of dissociation is 7 X 10(-10) M. The temperature stability and sulfhydryl dependence of the testicular androgen receptor are similar to androgen receptors in other organs. Binding is destroyed by heating the supernatants at 50 degrees C for 30 min and by exposure to p-chloromercuriphenylsulfonate (1 mM) at 0 degrees C for 60 min. Furthermore, like other androgen receptors, the half-time of dissociation of testicular androgen-receptor complexes at 0 degrees C is extremely slow (t1/2 greater than 35 h). Separation of seminiferous tubules from interstitial tissue showed that a major portion of these receptors were localized within the seminiferous tubules.     

Accumulation of carnitine in rat epididymis after injection of [3H]butyrobetaine in vivo: quantitative aspects, and the effects of androgens and antiandrogens

After i.m. injection of [3H]butyrobetaine into intact and castrated rats, the specific activity of plasma carnitine remained nearly constant over 24--96 h and epididymal uptake of carnitine was constant per unit time up to 72 h. The uptake ratio of intact to castrated rats was high at 48, 72 and 96 h after injection. Administration of estradiol valerate over 20 days reduced carnitine uptake in epididymis. This reduction was dose-dependent when estrogen was administered i.m. at 0.33--10 microgram/day levels. A maximum reduction of 90% was obtained with the 10 microgram dose. A dose increase from 33 to 100 microgram/day caused no further reduction. Norspiroxenone (2--10 mg/day) and SK 7670 (1.5 and 7.5 mg/day) were less effective than estradiol valerate (10 microgram/day) in suppressing carnitine uptake in epididymis. Epididymal carnitine uptake in estradiol valerate treated rats (33 microgram/day for 20 days) increased in a time- and dose-dependent manner under testosterone propionate treatment (50, 250, 1250 microgram/day). Carnitine uptake increased to 80% of the nonsuppressed levels when testosterone propionate was adminsitered over a 6-day period at 1250 microgram/day. Dihydrotestosterone increased epididymal carnitine uptake to the same extent as testosterone propionate. delta4-androstene-3,17-dione and 5alpha-androstane-3alpha,17beta-diol (50 microgram/day) were less effective, stimulating uptake to only 15% and 40% respectively of the testosterone propionate (250 microgram/day) stimulated levels. Changes in epididymal carnitine uptake evoked by various experimental procedures were closely paralleled by weight changes in the ventral prostate. This response resemblance indicates a similarity between the androgen sensitivity of the prostate gland and that of the carnitine uptake system in epididymis. The dose-dependent effect of estrogen on the accumulation of epididymal carnitine, together with the marked responses induced in this system by manipulation of its androgen status, support a possible use for the system as an assay for androgen or antiandrogen potency in vivo.     

Androgen-dependent accumulation of carnitine by rat epididymis after injection of [3H]butyrobetaine in vivo.

After i.m. injection of [3H]butyrobetaine into rats, the accumulation of carnitine into the epididymis, prostate gland, seminal vesicles, testis and heart was studied. The concentration of radiolabeled carnitine into the cauda epididymis increased linearly with time up to 72 h after the injection of the precursor, while its level in the prostate and seminal vesicles decreased rapidly. Very low levels of carnitine were found in the testis. Castration reduced the carnitine accumulation by cauda epididymis to 6% of the control levels while treatment of castrated animals with testosterone propionate (500 mug/day) partly restored the carnitine uptake. Similar treatment with 17beta-oestradiol valerate or 17alpha-hydroxyprogesterone had no effect. Surprisingly, cyproterone acetate (5 mg/day) also significantly stimulated carnitine accumulation by the epididymis to a level above that of the castrated controls. Simultaneous injection of both cyproterone acetate and testosterone propionate to castrated animals caused an additive effect of these steroids. This indicated that cyproterone acetate in this system is working as a weak androgen. Treatment of rats with 17beta-oestradiol valerate also reduced carnitine accumulation by the cauda epididymis. This is due to suppression of pituiatry gonadotrophin secretion, since concommitant treatment with testosterone propionate (500 mug/day) caused a normalization of the carnitine uptake. Treatment of intact rats with cyproterone acetate significantly reduced the epididymal weight, but not the carnitine accumulation. 17alpha-Hydroxyprogesterone treatment had no effect either on the epididymal weight or the accumulation of the carnitine. Unilateral orchiectomy reduced the carnitine accumulation by the cauda epididymis to about 40% of that occurring in the non-operated control side. This indicates that the luminal contact between the testis and epididymis or the luminal content of the epididymis itself is of importance for the androgen-dependent metabolic process occurring in the cauda epididymis. Castration or hormone treatment did not change the conversion of butyrobetaine to carnitine, or the carnitine uptake by heart. Carnitine uptake by the testis after [3H]butyrobetaine injection was rather low and this would exclude the possibility of synthesis of carnitine in the testis as a source of epididymal carnitine. Carnitine only accumulated in the cauda epididymis in vivo 4 to 96 h after injection of [3H]butyrobetaine. The presence of radioactively labeled butyrobetaine or methylcholine was not detected.     

Evidence that androgen-binding protein endocytosis in vitro is receptor mediated in principal cells of the rat epididymis

We have studied the binding of [125I-iodo]androgen-binding protein (ABP) and of [3H]delta 6-testosterone photoaffinity-labelled ABP to receptors in the plasma membrane of rat epididymal cells in three ways: ABP binding to a Triton X-100-solubilized membrane extract, ABP binding to isolated epithelial cells in suspension and autoradiography of segments of dissected epididymides after in-vitro intraluminal injection of labelled ABP. The binding of iodinated ABP to the receptor was similar to that of photoaffinity-labelled ABP in gel filtration. The ABP-receptor complex was eluted from Superose 6 gels as an aggregate, with a molecular mass of 2000 kDa. It was separated into two peaks by sucrose gradient ultracentrifugation, with respective sedimentation coefficients of 18.4 and 9.0 s. The activity of the receptor (ABP-binding capacity/mg protein) was tenfold higher in the caput than in the cauda. The binding of ABP to the receptor was pH dependent, being almost abolished at pH less than 4. The binding at 4 degrees C of photoaffinity-labelled ABP to epithelial cells corresponded to two types of binding sites. The numbers of high-affinity and low-affinity sites per cell were 1600 and 7700 respectively; the association constants of these sites were 67.9 and 2.8 litres/nM respectively. The binding was decreased by treatment of the cells with trypsin or incubation in the presence of EDTA. The binding in vitro of labelled ABP to the epididymis epithelium reached a maximum after about 20 min at 4 degrees C. In the autoradiographic study the tracer was found to be closely associated with coated pits, coated vesicles, endosomes and pale multivesicular bodies. Treatment of rats with cycloheximide significantly reduced the uptake of the tracer. Perfusion in vitro of epididymides with chloroquine produced a fourfold increase of the tracer in endosomes and multivesicular bodies.     

The presence of androgen-binding protein in the guinea-pig testis, epididymis and epididymal fluid

Androgen-binding protein (ABP) is present in the guinea-pig testis, epididymis and epididymal fluid. Guinea-pig ABP sediments as an approx. 4.6S species on sucrose gradients containing 0.01 M KCl. Electrophoresis on non-denaturing polyacrylamide gels indicated that specific androgen binding was present in epididymal cytosol, but not in plasma. Time-course studies indicated that binding equilibrium is approached in about 2.5 h; the dissociation half-time of [3H]5 alpha-DHT from guinea-pig ABP is 5.64 +/- 0.62 h (n = 6) at 4 degrees C. The relative affinities of some steroids for guinea-pig ABP in relation to 5 alpha-DHT = 1 are: testosterone = 0.55 +/- 0.13 (n = 4), estradiol = 0.14 +/- 0.03 (n = 4), the anti-androgen cyproterone acetate = 0.0025 +/- 0.0002 (n = 3). Guinea-pig ABP exhibited an equilibrium dissociation constant of 6.34 +/- 0.52 nM (n = 3) at 4 degrees C and there were 3.43 +/- 0.78 (n = 3) pmoles of binding sites per mg of protein when homogenates of the whole epididymis were assayed. The concentration of ABP was lowest in the caput-corpus region of the epididymis, highest in the proximal cauda, and intermediate in the distal cauda. Essentially all of the ABP present in the distal cauda was intraluminal, as evidenced by the fact that flushing of the duct eliminated most of the [3H]5 alpha-DHT binding activity.     

Glucocorticoid-induced cell-size changes and nuclear fragility in rat thymocytes

The events preceding glucocorticoid-induced lymphocytolysis have been studied in isolated rat thymocytes. Incubation of thymocytes at 37°C in the presence of 1 μM dexamethasone resulted in the progressive appearance of pyknotic cells of modal diameter 4.6 μm, distinct from normal cells of diameter 5.2 μm. The rate of appearance of the pyknotic cells was determined by selective electronic cell counting, and was shown to be accompanied by increased nuclear fragility. The production of pyknotic cells was glucocorticoid-specific, dose-dependent, blocked by cycloheximide, and preceded the loss of cell viability as determined by dye exclusion. The pyknotic cells were separated from the non-pyknotic cells by density-gradient centrifugation and shown to be solely responsible for the observed nuclear fragility.     

Prolongation of islet allograft and xenograft in nonimmunosuppressed rat recipients

The temperature at which islets were cultured have been observed in this study to play a major role in the prolongation of islet survival when subsequently allotransplanted across a major histocompatibility barrier. Culturing of Lewis (Le) and Wistar rat islets for 7 days at 26° prior to transplantation prolonged their functional period significantly in $\text{4}\text{6}$ and $\text{3}\text{5}$ of the nonimmunosuppressed diabetic ACI recipient animals. Monkey islets when cultured for 7 days at 26°C had significantly prolonged the functional period in $\text{2}\text{5}$ of the diabetic recipient Le rats. In contrast, in vitro culture for 7 days at 32°C or for 7–14 days at 37° had failed to prolong Le islet survival in the ACI recipient rats. Rejection of successful allografts of cultured rat islets was subsequently induced with an intravenous (i.v.) injection of donor splenocytes, suggesting that the successful allografts maintain their antigenicity. The immunogenicity of the islet allograft in provoking humoral response by the recipient was found to decrease following in vitro culture for 7 or more days regardless of the variation in culturing temperature. This was demonstrated by the lowered antidonor antibody titer in the recipients of cultured islets as compared with those that received fresh islets. Data in this investigation demonstrates that culture of islets under a relative low temperature (26°C) can prolong the survival of islet allograft or xenograft in the nonimmunosuppressed recipient, and alteration of humoral immunity alone cannot account for prolongation of islet allograft survival.     

Glucocorticoid receptor in human embryo fibroblasts. I. Kinetic and physicochemical properties.

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1--3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5--9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone--receptor complexes was observed.     

Relation of membrane property of microsomes to androgen biosynthesis

Spin-labelled fatty acids I(12,3) and I(1,14) were incorporated into microsomal membrane of cryptorchid mouse testis and Leydig cell tumor as well as liver. The freedom of motion of spin of I (12,3) was more restricted in testis microsome than in liver. At the lower temperatures, the freedom of motion of spin in the tumor microsomes was similar to that in the testis, but at higher temperature (20–50°C) was much greater. Plotting of the empirical parameter, h₀/h-_?1, calculated by the spectra of I(1,14), against the reciprocal of the absolute temperature clearly showed two inflection points in both liver and testis microsomes, one at 19°C and the other at 30°C. On the other hand, tumor microsomes lacked these break points and permitted spin to move more freely.These results suggest that tumor microsomes contain the increased fluidity. The importance of membrane fluidity in relation to steroid biosynthesis was also discussed.     

Characterization of a nuclear receptor for testosterone in seminiferous tubules of mature rat testes.

A specific androgen receptor could be demonstrated in the nuclear and cytoplasmic fractions of testicular tissue of mature hypophysectomized rats, either in vivo after injection of testosterone or in vitro after incubation of testis tissue with testosterone. Using agar-gel electrophoresis this receptor could be distinguished from the testicular transport-like protein for androgens (androgen binding protein = ABP). After in vivo administration of testosterone the steroid bound to the receptor in mature rat testis was mainly unmetabolized testosterone. After dissection of testis tissue the larger part of the receptor was shown to be present in the seminiferous tubules. The amount of exogenous testosterone that could be bound per mg of protein in the nuclear extract increased gradually during 20 days after hypophysectomy. Some characteristics of the receptor in the nuclear extract and of ABP were compared : the receptor was more sensitive to temperature increases than ABP; the steroid dissociated more slowly from the receptor than from ABP; cyproterone acetate showed almost no effect on the binding of dihydrotestosterone to ABP, but did compete for the receptor binding sites in the nuclear extract.     

Characterization of a cytoplasmic androgen receptor in the ram testis

An androgen receptor has been characterized in the cytosol fraction of testes from hypophysectomized adult rams after in vitro labelling with [3H]testosterone. It can be distinguished from the testicular androgen-binding protein (ABP) and from the plasma 5 alpha-dihydrotestosterone-binding protein by electrophoresis on 3.25% acrylamide gels (Rx = 0.5) and on agar gels (anodic migration). It sediments in the 4S region in sucrose gradient containing 0.4 M KCl. Its complex with testosterone dissociates very slowly (t 1/2 = 29 h at 0 degrees C), and is destroyed by heating at 50 degrees C for 30 min and by pronase. Its relative affinities for steroids are 5 alpha-DHT greater than T greater than 5 alpha-androstanediols greater than cyproterone acetate greater than estradiol greater than progesterone. The number of binding sites is limited (about 20 fmoles/mg protein) and the apparent equilibrium dissociation constant (KD) is 5 x 10(-9) M.     

Some properties of androgen-binding activity in rat testis.

High-affinity (Ka approximately equal to 5 X 10(8) M-1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant (t1/2 = 3 min, 0 degrees C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 degrees C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein (Rf 0.54) from albumin (Rf 0.62). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [3H]testosterone.     

Cortisol metabolism in lymphocytes from cancer-bearing patients

A study was conducted to determine the pattern of cortisol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1,2-³H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-bearing patients: 20α-hydroxycortisol, 20β-hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.     

Regulation of gonadal function in uremia

Gonadal function and its neuroendocrine control were studied in seven uremic men on chronic hemodialysis. Testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were measured in plasma or serum by established radioimmunoassay techniques. The metabolic clearance rate (MCR) of testosterone was also determined by constant infusion of ³H testosterone, and blood production rates were then estimated from the AM plasma level and the metabolic clearance rates. Predialysis, plasma testosterone in these uremic men were subnormal, measuring only $\text{289 ng}\text{100 ml ± 60 SE}$ (normal male values are 660 ± 70). The blood production rate of testosterone was also subnormal, averaging 2.7 mg/day in the two individuals studied, since the metabolic clearance rates were normal. However, after dialysis testosterone levels rose to $\text{408 ± 49 ng}\text{100 ml}$ (p < 0.01), the MCR to 1570 liters/day, and the calculated mean production rate increased to a normal level of 6.9 mg/day. Base-line serum LH was increased in the uremic state to 22.7 ± 1.8 mlU/ml (normal 5.9 ± 2), while FSH levels were normal at 7.7 mlU/ml ± 1.2 SE. Dialysis did not significantly alter serum immunoassayable gonadotropin levels. Gonadal stimulation by chorionic gonadotropin (4000 IU/day) initially did not increase testosterone levels, but continued stimulation-caused a two- to threefold rise by the fourth day. Clomiphene citrate (200 mg daily for 7 days) produced variable changes in testosterone and LH; two subjects did not respond, while two had a rise in both testosterone and gonadotropin. Luteinizing releasing hormone (LRH-Guillemin 150 μg intravenously) caused a two- to threefold rise in serum LH in both subjects studied. This work indicates that uremic men have subnormal testosterone production which is rapidly reversible with hemodialysis. The subnormal testosterone production appears to be primarily that of Leydig cell dysfunction, since there is elevated serum immunoassayable LH and a blunted initial response to exogenous gonadotropin.     

Nuclear accumulation of androgens in perfused rat accessory sex organs and testes.

The uptake of androgens into the nuclei of caput epididymis, ventral prostate, seminal vesicle and testis was studied by recirculating physiological and pharmacological concentrations of [3H]testosterone in an artificial medium through the lower half (hemicorpus) of castrated or hypophysectomized rats. The accumulation of dihydrotestosterone in accessory sex organ nuclei was saturable, inhibited by perfusion of excess testosterone or cyproterone acetate, and associated with binding to 3S salt-extractable molecules. In castrated preparations the mean saturation levels (pmol/mg DNA) were different in the three organs: seminal vesicle, 2.8; ventral prostate, 1.8; caput epididymis, 0.9. The saturation level was significantly lower in ventral prostate of hypophysectomized rats (1.2) treated with testosterone to regenerate the accessory sex organs. Testosterone was the major nuclear androgen in the testes of mature hypophysectomized preparations perfused with testosterone. Although there was a large amount of nonspecific accumulation, testosterone binding to 3S molecules was shown by sucrose gradient centrifugation. Binding of dihydrotestosterone to 3S molecules in testicular nuclei was also demonstrated. The ratio of dihydrotestosterone to testosterone was different in immature and mature testicular nuclei and was altered by treatments known to affect testicular 5 alpha-reductase activity. The results suggest that in rat accessory sex organs and immature testis the major active androgen is dihydrotestosterone, whereas in mature testis it is testosterone. The shift in the predominant nuclear androgen in the testis from dihydrotestosterone to testosterone is most simply explained by the maturational change in 5 alpha-reductase activity.     

High-affinity binding of the antiestrogen [3H]tamoxifen to the 8S estradiol receptor

The interaction of tamoxifen (ICI 46,474), a synthetic antiestrogen, with uterine cytosol proteins of immature calf and rat has been studied directly using the tritiated compound labeled with a high specific activity. The binding complexes were measured by the dextrancoated charcoal, protamine sulfate and hydroxyapatite assays. Scatchard plots revealed a single class of high-affinity (KD congruent to 1.7 nM) binding sites, with a binding capacity similar to that of estradiol. Competitive experiments showed the same binding specificity for estrogens and antiestrogens. Sucrose gradient analysis revealed an 8S binding protein which could be partially proteolysed by trypsin into a 4S binding protein. Kinetic studies showed that the association rate of tamoxifen was 5 times lower than that of estradiol and reacted according to a second order kinetics. The first-order kinetics of dissociation was considerably higher than that of estradiol, giving a half-dissociation time of 20--40 min at 0--2 degrees C. In some cases tamoxifen displayed two slopes of dissociation, but the proportion of the slow-dissociating complex was always inferior to that found with estradiol. In contrast to estradiol, the kinetic constants ratio (k-/k+) gave a calculated dissociation constant, similar to that determined in equilibrium conditions (KD), agreeing with a simple reactional scheme. We conclude that the antiestrogen tamoxifen binds directly to the 8S cytosol receptor for estrogens and not to another receptor for the antagonists. In contrast to estradiol, the antagonist is rapidly dissociated from the receptor sites and is unable to protect them against thermal inactivation. The affinity of tamoxifen for its receptor sites as determined directly is surprisingly high when compared to its affinity evaluated indirectly by competitive experiments. It is then suggested that the two ligands either bind on two different sites of the same protein or induce a different conformational change of the same binding site.     

Alcohol-induced cardiac hemodynamic and Ca2+ flux dysfunctions are reversible

Cardiac hemodynamic function and calcium accumulation by cardiac sarcoplasmic reticulum membrane vesicles were studied in control rats, in rats that had consumed alcohol as 39% of their daily calories for 10 months, and in alcoholic rats after a period of withdrawal from alcohol to determine if alcohol-induced alterations were reversible. Cardiac pump function was assessed with an isolated working whole heart preparation. Alcoholic rat hearts exhibited decreased response of left ventricular peak systolic pressure, $\text{d}\text{P}\text{d}\text{t}{}_{\mathrm{<mtext>max</mtext>}}$, peak aortic flow, and $\text{?}\text{d}\text{P}\text{d}\text{t}{}_{\mathrm{<mtext>max</mtext>}}$ to challenge with the sympathomimetic agent, dobutamine (0.1 μm). After alcohol was withdrawn from the diet for 4 to 6 months, cardiac hemodynamic response to dobutamine was not significantly different from controls. In alcoholic rats, Ca²⁺ binding and uptake were significantly lower than in controls. Four to six months following withdrawal of alcohol, Ca²⁺ binding and uptake had returned to control values. The data indicate that chronic alcohol consumption results in cardiac impairment characterized by inability of the heart to fully respond to β-adrenergic stimulation. This adrenergic subsensitivity may be caused, at least in part, by defect(s) in relaxation and excitation-contraction coupling mediated by intracellular Ca²⁺ fluxes. These defects were reversible since cardiac pump function and calcium accumulation returned to normal after withdrawing alcohol.     

Altered equilibrium between cortisol and cortisone in plasma in thyroid dysfunction and inflammatory diseases

The radioactivities of cortisol and cortisone in plasma were determined following simultaneous injection of ¹⁴C-cortisol and ³H-cortisone. The plasma concentrations of ¹⁴C-cortisol and ³H-cortisone decreased as a first-order function of time after an initial rapid drop, while there was a prompt appearance of ¹⁴C-cortisone and ³H-cortisol in plasma, which also decreased as a first-order function. The biologic half-lives of these four isotopic steroids were essentially identical. The ratio of ¹⁴C-cortisone to ¹⁴C-cortisol and that of ³H-cortisone to ³H-cortisol in plasma were constant after 60 min following injection and were identical, which suggested that cortisol and cortisone in plasma were at dynamic equilibrium. This ratio was 0.36 ± 0.01 (SE) in normals; it was decreased in patients with hypothyroidism (0.21 ± 0.03) and inflammatory diseases (0.18 ± 0.01) and was variable in hyperthyroid patients (0.42 ± 0.11). The ratio of the metabolic clearance rate of cortisone to that of cortisol was significantly increased in hypothyroid patients and in patients with inflammatory diseases, while urinary 11-ketonic metabolites of cortisol are known to decrease relative to its 11-hydroxy metabolites in these patients. These data and the decreased cortisone-to-cortisol ratio at equilibrium were consistent with the altered equilibrium between cortisol and cortisone, favoring cortisol, in these patients. It was suggested that the altered equilibrium between these steroids may be an important factor in determining the effectiveness of secreted or exogenously administered cortisol and the plasma concentration of cortisone in several disorders.