Biomedical subjects

D Bellabarba

Publications and source records attributed to D Bellabarba.

At least 37 records · Page 2Linked to original sources

PubMed1989

Ca2+, but not membrane lipid hydrolysis, mediates human chorionic gonadotropin production by luteinizing hormone-releasing hormone in human term placenta.

We postulated a role for lipid metabolism and Ca2+ in the LHRH-induced release of hCG by human placentas. Term placental cells in suspension prelabeled with [3H]myoinositol were stimulated without or with increasing concentrations of LHRH in the presence of 10 mM LiCl, and total inositol phosphate (IP) was measured by ion exchange chromatography; a nonsignificant 0.9 +/- 0.08-fold increase over the control value was observed. In contrast, placental cells stimulated with equimolar concentrations of angiotensin II (AII) induced a 4.6 +/- 0.9-fold increase in total IP (P less than 0.01), while rat pituitary cells showed 1.9 +/- 0.2- and a 2.4 +/- 0.07-fold increases in total IP production after stimulation with LHRH or AII, respectively (P less than 0.05). These increases were blocked by coincubation with specific LHRH and AII antagonists. When 1 x 10(6) placental cells were incubated with 45Ca2+ without and with increasing doses of LHRH for 0-75 s and then filtered under negative pressure, we observed significant incorporation of 45Ca2+. This influx was linear with incubation time, significantly more pronounced in cells exposed to LHRH than in control cells, and showed a dose-response curve to LHRH that reached maximal influx rates of 3.6 +/- 0.3 nM/min.1 x 10(6) cells with 10(-5) M LHRH. This response was completely blocked by coincubation with 10(-5) M LHRH antagonists; cobalt chloride and verapamil reduced it by 60% and 80%, respectively. Compared to placental cells stimulated with LHRH alone, those coincubated with LHRH and specific LHRH or Ca2+ antagonists released from 10-100% less hCG. We conclude that Ca2+ participates in the LHRH action in human placentas, but uncoupled to PI turnover.

Angiotensin II↗

PubMed1989

Angiotensin II stimulates both inositol phosphate production and human placental lactogen release from human trophoblastic cells.

We studied the functional significance of the binding of angiotensin-II (AII) to human placentas. Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue. Cell incubations with increasing doses of [125I](SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C. The results indicated the presence of specific low capacity [4300 +/- 1300 (+/- SE) sites/cell], high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for [125I](Sar1)AII. This binding was specific for AII analogs. When placental cells were preloaded with 40 microCi/mL [3H]myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production [EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L], as measured by ion exchange chromatography. (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L. AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII. AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion. The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production. These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown. The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.

Angiotensin II↗

PubMed1989

Basal and stimulated LHRH receptor sites in the pituitary of aging female mice.

We studied the regulation of pituitary luteinizing hormone-releasing hormone (LHRH) receptors in female mice aged 4-8 months (young), 10-14 months (middle-aged) and 15-18 months (old). In intact animals the mean +/- SE pituitary content of LHRH receptor sites ranged from 34.4 +/- 6.3 fmoles to 41.5 +/- 5.0 fmoles and remained constant throughout aging. Similarly, receptor affinity for LHRH was unaltered (range 1.0-4.0 X 10(9) M-1) by age. Ovariectomy (ovx) resulted in a 30-50% decrease in pituitary LHRH receptor sites (p less than 0.05) and the pattern of LHRH receptor fall after ovx was similar in all three age groups. Administration of estrogens (E2) at either a low (0.2 mg) or a high (2.0 mg) concentration to ovx mice prevented this fall in LHRH receptor sites and, in all three age groups, increased pituitary values to levels comparable to those measured in intact animals. We conclude that there are no significant differences in the basal and stimulated number or affinity of pituitary LHRH receptors between young, middle-aged and aged female mice.

Aging↗

PubMed1988

Aldosterone nuclear receptors in kidneys of chick embryo.

We have studied the properties of the nuclear receptors for aldosterone in kidneys of chick embryo. Aliquots of 0.4 M KCl nuclear extracts were incubated with [3H]aldosterone with or without 1 microM RU28362, a potent glucocorticoid analog. Scatchard analyses of binding data revealed two classes of binding sites with Ka of 0.26 and 0.03 X 10(9) M-1 and Nmax of 330 fmol and 620 fmol/mg DNA respectively. In presence of RU28362, however, we observed only a single class of binding sites with a Ka of 1.02 X 10(8) M-1 and a Nmax of 90 fmol/mg DNA. Competition studies performed in presence of RU28362 showed that aldosterone was the more effective competitor followed by corticosterone, progesterone, deoxycorticosterone, dexamethasone, cortisol, triamcinolone acetonide and cortisone. The nuclear complexes had a sedimentation coefficient in the area of 8 S which changed to 4-5 S in the presence of 0.4 M KCl. This effect of KCl was prevented by the addition of 10 mM sodium molybdate. Always in the presence of the glucocorticoid analog, by DEAE-c chromatography we observed a major specific aldosterone-binding fraction which was eluted with 0.2 M KCl. This fraction sedimented at 8.4 S in the absence of sodium molybdate and KCl. In the absence of RU28362, DNA-c columns retained only a small portion of the nuclear complexes which were eluted with KCl. These complexes sedimented, on sucrose gradient, at 4.6 and 3.1 S, whereas those which did not bind to DNA-c had a sedimentation coefficient of 8 S. In the presence of RU28362, the majority of bound [3H]aldosterone remained in the column flow-through fraction; when this fraction was further analyzed on DEAE-c, complexes were eluted with 0.2 and 0.3 M KCl. These data indicate that nuclear receptors for aldosterone are present in small number in kidneys of chick embryo and that they are mostly in the 8 S form.

Aldosterone↗

PubMed1988

Functional opioid receptor sites in human placentas.

We characterized the presence of opioid peptide receptor sites in plasma membranes and cells from human midterm and term placentas. Incubations with [3H]ethylketo-cyclazocine (EKC) at increasing doses revealed the presence of high affinity, low capacity, opioid peptide receptor-specific binding of the kappa-type. Scatchard analysis of the binding data showed, in the plasma membranes, linear plots at both stages of pregnancy with similar mean equilibrium association constants of 1.31 +/- 0.29 (+/- SE) X 10(9) mol/L-1 (n = 4) at midterm and 0.52 +/- 0.63 X 10(9) mol/L-1 at term (n = 4). In placental cells (n = 4) from term gestations, the binding plots were curvilinear; the first component had a Ka of 5.51 +/- 0.50 X 10(9) mol/L-1, and the second component had a Ka of 1.33 +/- 0.81 X 10(8) mol/L-1 (P less than 0.01). When standardized per mg tissue protein, the number of binding sites in plasma membranes increased from 13.8 +/- 9.8 fmol at midterm to 50.0 +/- 18.6 fmol at term (P less than 0.05). For term placental cells, the concentration of binding sites was 81.2 +/- 36.0 fmol for the high affinity sites and 713 +/- 390 fmol for the lower affinity sites. Specificity for the kappa-type of OPR was found based on the inability of mu- or delta-opioid peptides, as well as LHRH and TRH, to compete for [3H]EKC binding. Term placental cells incubated with various doses of opioid peptides had a 50% increase in placental lactogen production. The increase was significantly higher than controls only with kappa-agonists (P less than 0.05), maximal with 10(-9) mol/L EKC, and completely inhibited by 5 X 10(-6) mol/L naloxone. These results expand on previous data demonstrating the presence of opioid peptide receptor in placental plasma membranes and suggest a role for opioid peptides in regulating secretion of placental lactogen by placental cells.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1988

Human placental LHRH receptor: agonist and antagonist labeling produces differences in the size of the non-denatured, solubilized receptor.

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kav's indifferent from each other but significantly different from the Kav's of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Affinity Labels↗

PubMed1987

Glucocorticoid receptors in chick embryos: properties and ontogeny of the nuclear renal receptor.

A putative nuclear receptor for glucocorticoids was identified in the kidney of chick embryo. This receptor was a thermolabile protein which was readily digested by proteolytic enzymes. Its sedimentation coefficient on sucrose density gradient was 3.5S and its MW approximated, according to the Svedberg formula, at 98,500 Da. Binding assays, performed with crude or purified nuclei, and nuclear extracts showed that the latter preparation was the most suitable for the binding studies since it yielded a Bmax of 8-11% with a very low nonspecific binding (1% or less). Scatchard plots performed at various days of embryogenesis revealed a single class of binding sites with an association constant (Ka) of 0.12 +/- 0.06 X 10(9) M-1 (mean +/- SD; n = 5) and a maximal binding capacity (Nmax) that rose from 3.9 +/- 1.2 fmol/micrograms DNA at Day 13 of age to 13.2 +/- 2.2 fmol/micrograms DNA at Day 16 and then rapidly fell to 1.8 +/- 1.1 fmol/micrograms DNA before hatching (means +/- SD; n = 5). Competition studies with various steroids showed that only glucocorticoids and, to a lesser degree, progesterone had an affinity for the receptor. These results demonstrate that this nuclear-binding protein had physiochemical properties similar to those attributed to other glucocorticoid receptors in target cells.

Animals↗

PubMed1987

Enhanced inhibition of estrogen receptor nuclear binding in the uterus of aged mice.

We have studied why uteri from aging mice show a decrease nuclear concentration of estrogen receptors (UER). While 50-60% of available cytosolic UER from ovariectomized (OVX) mice aged 4-8 months, upon physiochemical activation, are able to bind either to DNA-cellulose or nuclear suspensions from young animals, only 20-30% of comparable concentrations of cytosolic UER from mice aged 15-18 months did so under identical experimental conditions. Nuclear dilutions with uterine cytosolic fractions from estrogen treated OVX mice prior to determination of [3H] UER binding sites in nuclear suspensions decreased the number of nuclear ER sites in both age groups. However, we observed that cytosols from aged animals showed a greater ability to prevent [3H]E2 binding to nuclear sites when compared to young ones (inhibition index: 0.286 +/- 0.013 (SE) vs. 0.137 +/- 0.025, P less than 0.05). These changes occur independently of protein concentration and result from dilution of a specific endogenous inhibitor of [3H]E2 binding to nuclear sites. The significance of these observed differences is discussed.

Aging↗

PubMed1987

Effect of ACTH on cholesterol and steroid synthesis in adrenocortical tissues.

Previous studies have established that under normal conditions, adrenal HMG-CoA reductase activity is higher in hamsters than in rats and humans. The hamster reductase activity follows a diurnal rhythm corresponding to that of plasma ACTH and glucocorticoids [Endocrinology 107 (1980) 215] but not to that of aldosterone. ACTH treatments to hamsters increased reductase activity after a latency of 60 min; this enhancement was prevented by cycloheximide [J. steroid Biochem. 24 (1986) 325]. Immunotitration and immunoblotting studies confirmed that ACTH caused an increase in reductase protein synthesis. In rats, long-term (1-9 days) and short-term (3 h) treatments with ACTH also induced increase in adrenal HMG-CoA reductase activity and reductase protein. In the presence of iodoacetamide and inhibitors of proteolytic enzyme, a main specific band of enzyme was evinced in the area of 102 +/- 6 kDaMr, by Western blotting, for both hamster homogenate and microsomal preparations (Endocrinology, 120 (1987]. Similarly Mr values were found with rat adrenal preparations. The concentration of mRNA, analyzed using the c-DNA pRed-10 coding for the Chinese hamster ovary reductase, was increased in adrenals of hamsters treated with ACTH. The reductase mRNA levels also fluctuated during the day in parallel with those of reductase activity and reductase protein. In conclusion, these results indicate that ACTH and other conditions inducing a change in hamster adrenal HMG-CoA reductase activity provoke parallel changes in reductase mRNA and reductase protein content. ACTH acts on the adrenal reductase of species synthesizing large as well as small quantities of cholesterol, thus indicating the general importance of this hormonal control.

Adrenal Cortex↗

PubMed1987

Dynamics of LHRH binding to human term placental cells from normal and anencephalic gestations.

In order to improve our knowledge on human placental hCG production, we studied the binding of an LHRH agonist (N-Ac-Pro1,D-Leu6)-LHRH to third trimester intact placental cells from normal and anencephalic fetuses. In normal pregnancies, specific and saturable binding was found for both LHRH and its analogs with two classes of binding sites. Association constants were 4.7 +/- 2.2 (mean +/- SEM) X 10(5) M-1 for the low affinity sites and 1.7 +/- 0.8 X 10(8) M-1 for the higher affinity sites (P less than 0.01), and the estimated number of sites was 1.71 +/- 0.52 nmol/mg of cell protein and 2.79 +/- 0.54 pmol/mg of cell protein, respectively. Preincubation with increasing concentrations of LHRH agonist induced a progressive decrease in specific binding sites and manifested by a reduction in hCG production which paralleled the concentration of the agonist in preincubation buffer. Studies with placental cells from three anencephalic fetuses showed a decreased binding capacity for LHRH and its agonist, when compared to normal trophoblastic cells, as well as a reduced capacity to produce hCG. Our results suggest that mechanisms dependent upon LHRH binding to its receptor are required for placental hCG production in normal pregnancies. Furthermore our investigation suggests a role for the endocrine feto-placental milieu in the manifestation of these placental LHRH binding sites.

Anencephaly↗

PubMed1987

Adrenocorticotropin and the time of day both influence the amount and activity of 3-hydroxy-3-methylglutaryl coenzyme-A reductase in the hamster adrenal.

We studied the effect of ACTH, alone or in combination with cycloheximide, on hamster adrenal 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase protein content (mass) and activity. Immunoblotting was performed on both homogenate and microsomal preparations, using a rabbit antirat liver reductase antibody and 125I-labeled protein A. A dose-dependent increase in HMG-CoA reductase protein content and activity was produced by ACTH. A time-course study revealed a latency of 60 min, followed by a significant increase at 120 and 180 min, in this response. At 180 min, microsomal reductase activity was significantly increased 4.7-fold, whereas the reductase protein content of microsomes and homogenate preparations was enhanced 4.4- and 3.1-fold, respectively. We calculated that only 40-50% of the reductase protein content was microsomal, indicating that more than one pool of the enzyme is present in adrenals. The coadministration of cycloheximide with ACTH not only prevented the hormonal effect on the protein content and activity of the reductase, but also produced, 180 min posttreatment, 58-69% and 65-86% decreases in reductase protein and activity, respectively. Also, both reductase activity and protein content fluctuated in parallel during the day. In the presence of proteolytic enzyme inhibitors and iodoacetamide, a major band in the area of 102K mol wt was evidenced by immunoblotting. These results indicate that ACTH and other conditions that provoke a change in adrenal HMG-CoA reductase activity also induce, in parallel, a change in the reductase protein content. The basic unit of the reductase has an apparent mol wt of about 102K.

Adrenal Glands↗

PubMed1986

On the control of HMG-CoA reductase, a key regulatory enzyme of adrenal cholesterol synthesis.

In this study we have determined the effect of ACTH on the activity of HMG-CoA reductase in microsomes of hamster adrenals. Cycloheximide was used to study the dependence of the increased enzyme activity by ACTH on de novo protein synthesis. Microsomes were prepared and preincubated with and without NaF and in the presence or absence of phosphorylase phosphatase in order to differentiate between expressed (McNaF) and total (McPP) activity. ACTH induced (after 120 and 180 min) significant increases in HMG-CoA reductase activity with a latent period of 60 min for both McNaF and McPP preparations. Cycloheximide alone decreased the activity of the reductase and the coadministration of cycloheximide + ACTH caused a greater loss of activity. Also, both treatments produced an accumulation of free cholesterol in adrenals suggesting an increased turnover of the reductase by these substances. Preincubation of microsomes at 37 degrees C enhanced per se HMG-CoA reductase activity, but the relative increase produced by ACTH treatments or endogenous ACTH remained essentially the same. In conclusion, under experimental conditions used, the enhancement of HMG-CoA reductase activity produced by ACTH seem to be due to increased enzyme synthesis.

Adrenal Cortex Hormones↗

PubMed1986

Decreased nuclear binding of estrogen receptors in the pituitary of mice: correlation with biological effects throughout aging.

We have determined the biological impact of our previously reported decreased activation of pituitary E2 receptors (PER) in mice. Young (4-8 months), middle-aged (10-14 months) and aged (15-18 months) female mice were studied either intact (metestrous) or gonadectomized (ovx) for 2 weeks prior to decapitation. Recombination studies using heat-treated cytosolic PER and enriched nuclear fractions of various age groups showed a markedly reduced ability for nuclear binding with advancing age, despite unchanged affinity of activated PER for nuclear acceptor sites. Cross incubation studies of heat-activated cytosolic PER with nuclei from mice of various age groups suggested that the activation defect followed the onset of anestrous whereas a reduced nuclear ability to support receptor binding preceded this manifestation. In parallel with these endocrine changes we observed a decrease in baseline cytosolic progesterone receptor (RcP) concentration and in the activity of the enzyme G6PDH in intact aging mice. Ovx induced a further decrease of both markers in young and middle-aged, but not in old mice, while E2 administration induced a decreased pituitary response in RcP but not in G6PDH in aged mice. Our results suggest functional changes in cytosolic nuclear interactions in the pituitary of aging female mice.

Aging↗

PubMed1986

On the role of luteinizing hormone-releasing hormone in the in vitro synthesis of bioactive human chorionic gonadotropin in human pregnancies.

The dynamics of luteinizing hormone-releasing hormone (LHRH) induced human chorionic gonadotropin (hCG) production were studied in isolated placental cells from normal and anencephalic midterm and term gestations. A spontaneous release of immunoreactive hCG was first detected after 24-36 h of preparation in term control cells. The addition of LHRH at a concentration ranging from 10(-9) to 10(-6) M induced a threefold increase in this output of hCG. Placental cell responsiveness to LHRH varied according to the number of days of cell cultures, with maximal response on days 1 and 6. Placental cells from normal pregnancies incubated with 1 X 10(-6) M LHRH showed a release of both immuno- and bio-assayable hCG, which was four- to six-fold higher at midgestation than at term (p less than 0.001). In contrast, placental cells from pregnancies with anencephalic fetuses showed, at both stages of gestation, an hCG production that was comparable to that observed with normal term placental cells. We conclude that LHRH at a concentration appropriate for its placental receptor binding affinity induces a production of bioactive hCG in humans. Furthermore, our data suggest that anencephaly changes the placental response of hCG to LHRH stimulation.

Anencephaly↗

PubMed1986

Effect of aging on the dissociation kinetics and estradiol receptor nuclear interactions in mouse uteri: correlation with biological effects.

The dissociation kinetics of uterine estradiol receptor (UER) from young, middle-aged, and old mice have been studied in vitro and correlated with interactions of the steroid receptor with nuclear suspensions in a cell-free system. Furthermore, we have determined in these various age groups of mice the concentration of uterine cytosolic progesterone receptors and the activity of glucose-6-phosphate dehydrogenase. Compared to UER from young mice, the receptor from middle-age and old mice displayed similar first order dissociation kinetics, but a significantly reduced fraction of the slow component resulting from transformation of the receptor into a state of higher affinity for estradiol. In all age groups, sodium molybdate markedly inhibited this heat activation of UER. Recombination studies using heat-treated cytosolic UER and enriched nuclear fractions of various age groups suggested a markedly reduced ability for nuclear binding with advancing age, despite unchanged affinity of activated UER for nuclear acceptor sites that ranged from 3-5 X 10(9) M-1. Cross-incubation studies of heat-activated cytosolic UER with nuclei from young, middle-aged, and old mice suggested that the activation defect of cytosolic UER was already present at middle age and that a reduced nuclear ability to support receptor binding followed the onset of reproductive acyclicity. In parallel with these endocrine defects, we observed, with aging, decreases in mean baseline progesterone receptor concentrations and glucose-6-phosphate activity in uteri of both intact and castrated mice. The diminution of these two parameters was present at middle age, further increased at old age, and persisted after administration of low (0.2 mg) or high (2.0 mg) doses of estradiol for 3 days before study. Our observations suggest that decreased receptor activation is primarily responsible for the decreased effects of estrogen in aging mice.

Aging↗

PubMed1985

Age-dependent, ovary-independent decrease in the nuclear binding kinetics of estrogen receptors in the brain of the C57BL/6J mouse.

To further define the role of aging of the brain in the induction of reproductive acyclicity, we put to death intact as well as castrated female C57BL/6J mice of various ages before and from 0.5 to 24 hours after subcutaneous injection of 0.2 micrograms of 17 beta-estradiol. Pooled hypothalamic and pituitary tissues were dissected and cytosolic/nuclear estrogen receptors were assayed in buffer that consisted of 10 mmol/L Tris(hydroxymethylaminomethane) hydrochloride, 1.5 mmol/L ethylenediaminetetraacetic acid, and 0.5 mmol/L dithiothreitol and contained molybdate (25 mmol/L) and inhibitors of proteases. Our results in intact animals indicated that baseline cytosolic concentration of estrogen receptors remained constant at 60 to 77 fmol/mg of protein (range) throughout aging, whereas nuclear levels of estrogen receptors decreased from 1.2 to 1.6 fmol/micrograms of deoxyribonucleic acid (range) to nondetectable levels after the onset of ovarian acyclicity. No age-related changes in the Ka were observed. After subcutaneous challenge with estrogen, nuclear binding of hypothalamic-pituitary axis estrogen receptors revealed a significant age-related decrease which was already evident at 10 to 14 months of age and prior to the onset of anestrous. Castration, whether performed neonatally or at 8 months of age, reduced the hypothalamic-pituitary axis concentration of estrogen receptors in middle-aged and aged animals, but did not prevent this blunted kinetics of nuclear binding. One week of daily injection of 17 beta-estradiol to intact and castrated mice of all age groups prior to binding kinetic studies induced maximal (five fold) increases in the content of hypothalamic-pituitary axis estrogen receptors in young animals which readily bound to the nucleus. After similar therapy to middle-aged and aged mice, minimal changes or even no changes were observed in both cellular estrogen receptor contents, despite similar increments in plasma levels of estrogen. These findings suggest an age-dependent decrease in the kinetics of hypothalamic-pituitary axis estrogen receptors manifested by a reduced synthesis of functional estrogen receptors in the brain of mice.

Aging↗

PubMed1985

Binding of thyroid hormones by nuclei of target tissues during the chick embryo development.

In the present studies we have compared the ontogeny of the binding of thyroxine (T4) and triiodothyronine (T3) to isolated nuclei from various target tissues of chick embryo. We observed a marked difference between the patterns of Satchard plots, maximal binding capacities (MBC) and association constants (Ka) of T4 and those of T3. Scatchard plots revealed that T4 and T3 had different binding sites. In liver, brain and lung MBCs and Kas of T3 and T4 were rather similar at day 9, but during the following days (12-19) T3 MBCs and Kas showed small changes, whereas T4 MBC markedly increased (4-5-fold) and T4 Ka significantly declined. In liver, for instance, T3 MBC = 395 +/- 19 (day 9) and 489 +/- 66 fmol/mg protein (day 19); T4 MBC = 631 +/- 6.5 (day 9) and 2201 +/- 516 fmol/mg protein (day 19); T4 Ka = 1.92 +/- 0.01 (day 9) and 0.56 +/- 0.21 X 10(8) M-1 (day 19). These data indicate that, during chick embryogenesis, nuclei of target tissues contain multiple T4 binding proteins, but only a single T3 binding site.

Animals↗

PubMed1985

On the presence of nonfunctional uterine estrogen receptors in middle-aged and old C57BL/6J mice.

The nuclear binding kinetics of uterine 17 beta-estradiol (E2) receptors (UER) were studied throughout aging in intact and castrated (OVX) mice. When compared to young animals, 15- to 18-month-old mice showed a significant reduction in their total cytosolic (0.526 vs. 0.405 pmol/uterus; P less than 0.05) and nuclear (0.37 vs. 0.16 pmol/uterus; P less than 0.01) UER content, whereas the affinity (Ka) for estrogens remained constant (0.8-1.6 X 10(9) M-1). This age-related decrease in UER was preceded by a blunted and retarded nuclear binding of UER at 10-14 months of age, which was further accentuated after transition from perimenopause. Ovariectomy (OVX), whether performed neonatally or in adulthood, reduced the total concentration of cytosolic and nuclear UER in each age group studied, but did not prevent this reduced nuclear binding observed in middleaged mice. However, when standardized per tissue protein, the mean number of cytosolic UER from young and middle-aged, but not old, mice was reduced by 50% after neonatal OVX (176.5, 178.4, and 218.8 fmol/mg protein, respectively), whereas it remained unchanged when OVX was performed in adulthood and the animals subsequently studied at peri- and postmenopausal ages (326.3 and 283.3 fmol/mg protein, respectively). Daily administration of a physiological dose of E2 for 7 days to OVX mice of each age group induced maximal synthesis of UER in young animals, but not in peri- and postmenopausal ones; in peri- and postmenopausal animals, this was paralleled by reduced uterotropic responses despite similar increments in plasma E2. These results suggest an age-related, gonad-independent decline in the number of functional UER early in reproductive aging.

Aging↗