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This study compares the functional consequences of levothyroxine (T4) treatment during pregnancy as well as the acute affects of triiodothyronine (T3) on spontaneous uterine contractile activities observed in vitro. Uterine biopsies were obtained from consenting women undergoing elective caesarean at term (n = 28). Spontaneous contractile activities from T4-treated pregnant women (n = 8) were compared with control patients (n = 20) by isometric tension measurements. Effects of acute T3 and T4 on control tissues were also monitored. Area under the curve, amplitude, time to peak, duration, and frequency were quantified. In uterine strips from women treated for hypothyroidism, phasic uterine contractions of larger amplitude (+77%) were observed, with a prolonged duration at 90% relaxation (+138%) and reduced frequency (-55%) compared with values of the control group. The addition of exogenous T3 in vitro on control strips induced a significant increase in the duration of the contractions and a significant decrease in frequency (P < 0.05), which partially mimics the results obtained in strips from T4-treated women. Significant modifications of contractile properties were observed in strips from pregnant women treated with levothyroxine, consistent with those observed with the addition of exogenous T3. Clinical practices of modern obstetrics should take into account the effect of thyroid hormones on uterine contractions' time course to ensure a tighter followup at the end of pregnancy to achieve safer delivery.

Background: Obesity is a worldwide health problem assuming epidemic proportions. Development of effective clinical interventions is needed to lower the impact of associated morbidity and mortality, without forgetting related costs. We have established an interdisciplinary clinic for obesity management, Unité d'Enseignement, de Traitement et de Recherche sur l'Obésité (UETRO), which consists of individual consultations combined with group sessions. We report here the effectiveness of this program for weight reduction over the first year of follow up., Methods: We performed retrospective analysis of standardized patient records of the first 115 consecutive subjects referred to UETRO with available follow up for 1 year., Results: Mean age, body mass index (BMI), and waist circumference (WC) of our cohort were 46 +/- 13 years, 44.7 +/- 0.9 kg/m(2), and 120.5 +/- 1.9 cm, respectively. Hypertension and diabetes were present in 46% and 23% of our patients. Weight and WC loss were gradual over 1 year and were significantly reduced by 6.6 +/- 0.8 kg and 6.7 +/- 0.7 cm, respectively (P < 0.001), without attainment of a plateau. Blood pressure and lipid profile significantly improved after 1 year of follow up. However, the proportion of patients taking metformin, lipid-lowering, antihypertensive, or antiobesity drugs increased significantly over follow up, reflecting intensification of treatment of co-morbidities and weight management. Significant weight and WC loss occurred independently of diabetes status and use of antiobesity medications., Conclusions: This program appears to be as effective for treating obesity as more intensive treatment programs. Future prospective studies are needed to evaluate the benefits and costs of this therapeutic approach.

In obesity, it is well known that basal growth hormone (GH) levels can be suppressed and they may show an impaired response to various stimuli like hypoglycemia, stress, and GHRH. However, the inhibitory effect of hyperglycemia on GH in this condition has not been well studied. We analyzed the GH response to an oral glucose tolerance test (OGTT) in 25 unselected patients with morbid obesity who were not diabetic, glucose intolerant or affected by renal or hepatic disease. Ten (40%) of the 25 subjects had an abnormal response of GH to the OGTT, expressed by a lack of suppression of GH levels below 2 micrograms/L within 60 minutes of glucose administration. Three subjects even had a paradoxal increase of GH levels of more than 50% of the basal level. There was no significant difference between these subjects regarding their age, BMI and, serum levels of glucose, insulin, C peptide, or insulin/glucose ratio. After weight loss, three of four patients normalized their GH response. Thus, we conclude that patients with morbid obesity frequently have an abnormal response of GH to OGTT (40% vs 4-8% in normal subjects). This finding must be taken into consideration when interpreting GH levels in these patients. A normalization of this response can be expected after weight loss.

A 63-year-old female patient was referred to our hospital in February 1994 for a pituitary tumor. On a previous examination, in 1973, she had a goiter, nonspecific symptoms and only an elevated serum T3. In 1984 she had become hypothyroid, her goiter had increased, serum T4 was 69 nmol/L, TSH 34.4 mU/L, and TPO antibodies were positive. Hypothyroidism due to autoimmune thyroiditis was diagnosed and she received L-T4 100 micrograms/day. In 1985 and 1986, serum TSH had decreased but remained slightly elevated, while T4 was at the upper limits of normal. From 1987 to 1989 her serum TSH rose from 9 to 20 mU/L and remained at that level for the ensuing 4 years in spite of increasing L-T4 up to 150 micrograms/day. In October 1993, after discontinuing L-T4 for 6 weeks, TSH was 23.7 mU/L, T4 170 nmol/L, 131I thyroid uptake 52%, and the CT scan showed a large pituitary tumor with suprasellar extension. On preoperative investigation TSH was 40-51 mU/L with no response to TRH or GnRH. The alpha-subunit was increased at 6.33 micrograms/L with the alpha-TSH/TSH molar ratio of 1.23. Prolactin was elevated, but plasma cortisol, FSH, and LH were low. At surgery, we found a large chromophobe adenoma with few PAS-positive granules and with immunostaining positive for TSH and prolactin. From the clinical and biological data, we can conclude that the patient had probably a TSH-secreting adenoma since the goiter was first detected. The development, however, of autoimmune thyroiditis with hypothyroidism considerably modified the presentation of the disease and may have accelerated the growth of the tumor.

We previously reported that dopamine (DA) inhibited the release of human placental lactogen (hPL) from human placental cells. We also demonstrated the presence of D2-dopamine receptors in membrane preparations of human term placenta. The aim of the present study was to characterize these D2 receptors on freshly isolated human trophoblastic cells. The binding of [3H]-spiperone to these cells showed a curvilinear Scatchard plot suggesting the presence of two classes of binding sites (Kd1 = 1.26nM; Kd2 = 44.3nM). Competition experiments showed the following inhibitory binding potencies: serotonin-2 (5-HT2) > or = D2 >>> alpha-adrenergic, beta-adrenergic, D1-dopamine, thus suggesting the presence of 5-HT2 binding sites. We have examined this possibility by blocking [3H]-spiperone binding to 5-HT2 receptors in the presence of 50nM ketanserin, a selective antagonist of 5-HT2 sites. Under this condition, the linear Scatchard plot obtained suggested a single population of homogeneous binding sites for [3H]-spiperone with a Kd of 0.55nM. To further characterize placental D2 receptors we conducted binding experiments with [3H]-raclopride, an more selective D2 antagonist. The linear Scatchard plot obtained with this ligand suggested one class of binding sites for [3H]-raclopride (Kd = 6nM) with the following inhibitory potencies: D2 >>> beta-adrenergic >> 5-HT2, D1, alpha-adrenergic. These results suggest an important paracrine function for DA in human placenta and show for the first time that [3H]-spiperone binds putative 5-HT2 receptors in human placenta.

In this study we have demonstrated the presence of specific 3,5,3'-L-triiodothyronine (T3) binding sites in the synaptosomes of chick embryo cerebral cortex and described their ontogeny. Scatchard analyses of binding data obtained with synaptosomal preparations from 17-day-old embryos revealed two T3 binding sites. The first site (N1) had a high affinity and low capacity since its dissociation constant (Kd) was 68 +/- 1.3 nM T3 (mean +/- S.D.; n = 3-5) and its maximal binding capacity (Bmax) was 8.63 +/- 1.59 ng T3/mg of protein, whereas the second site (N2) had a higher Kd of 5.04 +/- 0.5 microM T3 and a larger Bmax of 405 +/- 49 ng T3/mg of protein. The relative affinity of the synaptosomal fraction for T3 and other analogs was the following: T3 greater than T4 (thyroxine) greater than D-T3 (3,5,3'-D-triiodothyronine) = TRIAC (triiodothyroacetic acid) greater than rT3 (reverse T3). Gel chromatography of the [125I]T3 labeled fraction revealed a partially saturable peak with an estimated MW of more than 100 kDa. The ontogenic pattern showed a progressive increase of Kd and Bmax of N1, occurring mainly between the 12 and 19 days of incubation, and a marked fall, particularly of the Bmax, after hatching. The second site did not show any important variation during the embryogenesis. These data indicate the existence of specific T3 binding sites in synaptosomes from cerebral cortex of chick embryo, whose properties and ontogeny are completely different from those of the nuclear receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effects of age on the roles of phosphoinositide (PI) and protein kinase C (PKC) in luteinizing hormone (LH) release by gonadotropin-releasing hormone from mouse pituitaries. Pituitary cells from intact and 14-day ovariectomized (OVX) mice aged 4-8 months, 10-12 months and 14-18 months were cultured at a dilution of 3 x 10(5) cells/ml of M199-bovine serum albumin medium for 3 days prior to stimulation with either buserelin or phorbol ester (phorbol myristate acetate, PMA), while LH was assayed by radioimmunoassay using anti-rat LH antibody (NIDDK-5-10). In intact young mice, buserelin and PMA specifically induced time- and dose-dependent increases in LH release with specific mean ED50 of 0.82 x 10(-11) M (buserelin) and of 1.6 x 10(-8) M (PMA) and a maximal LH release of 138 +/- 15 ng/10(6) cells after a 3 h stimulation period. Age did not affect the ED50 of either agonist but significantly reduced their ability to release LH. This reduction was more pronounced for buserelin than for PMA and was evident as early as middle-age. OVX resulted in a significant increase in both basal and stimulated LH release, but did not affect the age-related reduced secretion rate of LH by either agonist. Buserelin stimulated the incorporation of [3H]inositol into [3H]inositol phosphates (IP) in a dose-dependent manner, which was unaffected by either age or OVX. We conclude that, with aging, there occurs a reduced LH release rate to both buserelin and PKC stimulations, uncoupled to changes in PI-IP cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

In isolated human trophoblastic cells, dopamine (DA) significantly inhibited the angiotensin-II (AII)-stimulated inositol phosphate (IP) accumulation by 44 +/- 8% (EC50, 0.5 +/- 0.2 microM) and human placental lactogen (hPL) release by 85 +/- 5% (EC50, 1.0 +/- 0.8 microM). These effects were blocked by sulpiride, a specific D2 antagonist. On the contrary, scherring 23390 (a specific D1 antagonist) and propranolol (a specific beta-adrenergic antagonist) were ineffective, suggesting that these DA effects are mediated through a DA receptor of the D2 subtype. The mechanism by which DA inhibited AII-stimulated inositol phosphate production implicates a GTP-binding protein sensitive to the islet-activating-protein (IAP), since DA's effects on IP accumulation and hPL release were blocked by this toxin. To further characterize this GTP-binding protein, particulate fractions of placental cells were incubated with [alpha-32P]NAD and IAP. Solubilized extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins of 40 and 41 kDa mol wt were specifically ADP ribosylated. They were probably involved in the DA inhibitory processes, since IAP treatment, known to suppress the effects of DA, also reduced the labeling of these two molecules by around 40%. The effects of AII and DA on hPL release appear to be insensitive to the external calcium concentration, since the results were not significantly different in normal (1.8 mM Ca2+) and low Ca2+ (10(-5) M Ca2+) concentrations. On the other hand, increasing the intracellular concentration of cAMP by adding forskolin did not modify the effect of DA on either IP accumulation or hPL release, suggesting that cAMP is not implicated in hPL release from freshly isolated human trophoblastic cells.

We correlated the content of hypothalamic (HT) GnRH and pituitary (PT) GnRH receptor sites with PT and plasma gonadotropin levels throughout aging in C57BL/6J mice. Female mice of 4-6 months (young), 10-12 months (middle-age) or 15-18 months (old) of age were studied either intact or 15 days post-ovariectomy (OVX) with or without E2 therapy. In intact mice, HT GnRH content increased twofold during aging while GnRH receptor sites in PT remained unchanged. PT content of both FSH and LH gradually rose during aging while plasma concentration rose even more. OVX resulted in a significant decrease in both HT GnRH content and PT receptor sites and no age difference was observed. OVX also resulted in a significant increase in both PT content and plasma levels of gonadotropin in young and middle-age mice while old mice showed a blunted response. After E2 therapy for 7 days, HT GnRH content and PT GnRH receptor sites returned to normal levels in all age groups. By contrast, E2 therapy resulted in no change in PT content of FSH:LH in any age group. Whereas plasma FSH:LH levels returned to intact levels in young mice, they remained elevated to OVX levels in middle-age and old ones. Our results demonstrate an age related dichotomy in the PT production of FSH:LH unrelated to changes in either HT GnRH content or its PT receptor sites, thus suggesting cellular defects in post-receptor binding events within the pituitary.

4-Aminopyrazolopyrimidine (4-APP) treatments to rats for 3 days induced 2-fold increase of circulating ACTH and 11-fold increase of adrenal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA compared to NaCl-treated controls. This in vivo model was used to study the effect of the suppression of ACTH secretion on the adrenal HMG-CoA reductase mRNA level. Dexamethasone (Dex) administration to 4-APP-treated rats caused a rapid and parallel decline of the levels of plasma ACTH and adrenal HMG-CoA reductase mRNA to 50% within 2.5 h, whereas the free and esterified cholesterol content was increased 5 and 9.4 times respectively. These changes could be counteracted by the co-administration of ACTH with Dex. Aminoglutethimide (AG) administration to 4-APP-treated rats, which increased the adrenal esterified cholesterol content (7.5 times), decreased the HMG-CoA reductase mRNA level (44%), despite plasma ACTH level remaining elevated. Moreover, the participation of newly synthesized protein(s) in the lowering of adrenal HMG-CoA reductase mRNA level induced by ACTH suppression is suggested by the fact that cycloheximide (Cyclo), when co-administered with AG, completely blocked the decrease of HMG-CoA reductase mRNA level, despite the plasma ACTH level decreasing by 68% and the free and esterified cholesterol content increasing 3.9 and 12.3 times, compared to 4-APP-treated rats. Furthermore, the specificity of these effects was established by the fact that the beta-actin mRNA level was not affected by the administration of either Dex, AG, Cyclo, or AG + Cyclo to 4-APP-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The synthesis of hepatic cytosol malate dehydrogenase (MDH) can be stimulated by thyroid hormones under various conditions, and consequently, this enzyme has been considered as a marker of the expression of thyroid hormone action. Since the concentrations of serum thyroid hormones increase considerably during the late phase of chick embryogenesis, we have studied the relationship between this phenomenon and liver MDH activity. A delay of 5 days was observed between the increase in circulating T4 and T3 concentrations and the enhancement of MDH activity. However, treatment with pharmacological doses of T4 (60 microgram/day) resulted in increased MDH activity 48 h after administration of the hormone, while hypothyroidism induced by methimazole decreased the level of the enzyme. Sex-linked differences in MDH activity were found in newborn chickens between 5-9 days of age. No such differences were observed for serum T3 and T4. These findings suggest that under physiological conditions MDH activity is not directly modulated by thyroid hormones in the chick embryo. However, these hormones may well play a permissive role with this enzyme.

In this study we have examined the effect of neonatal hypothyroidism and triiodothyronine (T3) replacement therapy on the maximal binding capacity (MBC) and the affinity of T3 receptors prepared from liver, brain and lungs. Rats were radiothyroidectomized at birth and administered T3 or its placebo starting at 8 days of age. The thyroid state in normal, hypothyroid and hypothyroid T3-treated rats was assessed by serum determination of thyroxine, T3 and by the measurement of the hepatic alpha-glycerophosphate dehydrogenase. The animals were sacrificed at 8, 16 or 24 days of age and the T3 binding was estimated in isolated nuclei and in salt nuclear extracts. The MBC of the T3 receptors was higher in the hypothyroid rats at all ages when it was determined in isolated nuclei, but not when was measured in nuclear extracts. At 8 days, the MBC had risen twofold or more in the receptors from brain (1.0 +/- 0.37 vs. 0.4 +/- 0.1 ng T3/mg DNA in controls) and from lungs (0.6 +/- 0.25 vs. 0.3 +/- 0.15 ng T3/mg DNA in controls), but was only slightly elevated in the hepatic receptor (0.62 +/- 0.08 vs. 0.48 +/- 0.15 ng T3/mg DNA in controls). At 16-24 days, the highest value of MBC was observed in the hepatic receptor (0.83 +/- 0.04 vs. 0.41 +/- 0.1 ng T3/mg DNA in controls) followed by the brain receptor (0.65 +/- 0.03 vs. 0.35 +/- 0.02 ng T3/mg DNA in controls), and that of lung (0.39 +/- 0.07 vs. 0.20 +/- 0.03 ng T3/mg DNA in controls).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

An aldosterone receptor in the cytosol from kidney of chick embryos which had a sedimentation coefficient of 8.2 S and a molecular weight higher than 100,000 was identified. Kinetic analysis at 4 degrees revealed a rapid association of the hormone to the receptor that followed second-order reaction kinetics and a dissociation of pseudo-first-order reaction kinetics. The association (ka) and dissociation (kd) rate constants were, respectively, 4.94 X 10(5) M-1 sec-1 and 8.33 X 10(-6) sec-1. From their ratio a KA value of 5.9 X 10(10) M-1 was calculated. In a series of experiments performed with kidneys of 17-day-old embryos, the KA at equilibrium, obtained from the Scatchard plot, was 3.1 +/- 1.2 X 10(8) M-1, whereas the Nmax was 172 +/- 14 fmol/mg protein. Competition studies with various steroids demonstrated that corticosterone had an affinity for the receptor close to that of aldosterone, thus suggesting a degree of resemblance of the mineralo- and glucocorticoid receptors in the chick embryo. However, the profiles of the binding affinities and capacities during the embryogenesis showed that the aldosterone-binding sites had a pattern completely different from that of the glucocorticoid receptor, indicating that the two receptors are most likely separate entities.

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.

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.

Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.