Quantitative encapsulation and retention of 227Th and decay daughters in core-shell lanthanum phosphate nanoparticles.

Targeted alpha therapy (TAT) offers great promise for treating recalcitrant tumors and micrometastatic cancers. One drawback of TAT is the potential damage to normal tissues and organs due to the relocation of decay daughters from the treatment site. The present study evaluates La(227Th)PO4 core (C) and core +2 shells (C2S) nanoparticles (NPs) as a delivery platform of 227Th to minimize systemic distribution of decay daughters, 223Ra and 211Pb. In vitro retention of decay daughters within La(227Th)PO4 C NPs was influenced by the concentration of reagents used during synthesis, in which the leakage of 223Ra was between 0.4 ± 0.2% and 20.3 ± 1.1% in deionized water. Deposition of two nonradioactive LaPO4 shells onto La(227Th)PO4 C NPs increased the retention of decay daughters to >99.75%. The toxicity of the nonradioactive LaPO4 C and C2S NP delivery platforms was examined in a mammalian breast cancer cell line, BT-474. No significant decrease in cell viability was observed for a monolayer of BT-474 cells for NP concentrations below 233.9 µg mL-1, however cell viability decreased below 60% when BT-474 spheroids were incubated with either LaPO4 C or C2S NPs at concentrations exceeding 29.2 µg mL-1. La(227Th)PO4 C2S NPs exhibit a high encapsulation and in vitro retention of radionuclides with limited contribution to cellular cytotoxicity for TAT applications.

Covalent labeling of rat thymocyte and human lymphoid glucocorticoid receptor.

Lymphoid cells contain specific receptors for glucocorticoids. We have used [3H]dexamethasone-21-mesylate to label covalently glucocorticoid receptors in rat thymic lymphocytes and in neoplastic cells obtained from patients with acute lymphoblastic leukemia and malignant lymphoma. The covalently labeled glucocorticoid receptors were identified by polyacrylamide gel electrophoresis (in the presence of 0.1% sodium dodecyl sulfate). In cytosolic fractions prepared from rat thymic lymphocytes, [3H]-dexamethasone-21-mesylate labels a protein (Mr approximately equal to 95,000) which was identified as the glucocorticoid receptor by the following criteria: (a) labeling of this moiety is inhibited by treatment with a 100-fold molar excess of glucocorticoids, such as dexamethasone and triamcinolone acetonide; and (b) the covalently labeled Mr approximately equal to 95,000 protein is activated (by heating at 20 degrees for 30 min) to a form that binds to DNA-cellulose. When intact thymocytes are treated with [3H]dexamethasone-21-mesylate, an Mr approximately equal to 95,000 moiety is also labeled covalently. Approximately 35% of the glucocorticoid receptors can be labeled covalently when intact thymocytes are treated with 100 nM [3H]dexamethasone-21-mesylate for 30 min at 4 degrees. Neoplastic cells from acute lymphoblastic leukemia and malignant lymphoma were treated with [3H]dexamethasone-21-mesylate. In all samples, an Mr approximately equal to 95,000 moiety was labeled covalently; labeling was inhibited by excess glucocorticoid. Smaller moieties were also identified by competition experiments; these may represent proteolytic fragments of the Mr approximately equal to 95,000 receptor. Thus, in rat and human lymphoid cells, [3H]dexamethasone-21-mesylate can be used to label covalently the glucocorticoid receptor.

Effects of human growth hormone on insulin-stimulated glucose metabolism in 3T3-F442A adipocytes.

GH is believed to play a role in promoting insulin resistance in patients with diabetes and with GH excess. The means by which GH produces insulin resistance may be through direct suppression of glucose metabolism in target cells (insulin-independent) or by interfering with the ability of insulin to stimulate glucose metabolism (insulin-dependent). In 3T3-F442A adipocytes, long term incubation (24-72 h) with GH directly inhibits glucose oxidation and lipid synthesis in the absence of insulin. To distinguish the insulin-independent effects of GH on glucose metabolism from the insulin-dependent effects of GH, we examined the effect of GH on insulin-stimulated lipid accumulation in cultured 3T3-F442A adipocytes. Cells were incubated for 48-72 h with GH and then treated with insulin. Insulin stimulated lipid accumulation in GH-pretreated and control cells. Compared to control, GH-treated cells had lower absolute levels of lipid accumulation in the absence of insulin and at each insulin concentration tested. Thus, GH directly suppresses basal lipid accumulation and lowers the response to insulin. In addition, a 10 times higher insulin concentration was required to reach maximum stimulation of lipid accumulation in GH-treated cells (50 ng/ml) than in control cells (5 ng/ml). When cells were exposed simultaneously to insulin and GH for 72 h, GH treatment inhibited the ability of insulin to stimulate lipid accumulation, and the degree of suppression by GH was related to the GH concentration present. These observations suggest that GH suppresses glucose metabolism not only in the absence but also in the presence of insulin. Since short term (4-h) incubation with GH increases glucose metabolism transiently in GH-deficient preparations, we also examined the influence of short term incubation with GH on insulin responses. Cells were incubated for 4 h with varying concentrations of insulin in the simultaneous presence or absence of GH. Insulin stimulated the conversion of glucose to lipid when tested alone or in the presence of GH. Short term exposure to GH alone also stimulated glucose metabolism. The stimulation of lipid accumulation at insulin concentrations less than 5 ng/ml was greater with GH, but responses were comparable above 5 ng/ml insulin. The ability of insulin to bind to its receptor was not affected by prior treatment with GH for either short or prolonged time periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Bioactivity of human growth hormone in serum: validation of an in vitro bioassay.

GH, in clinical practice, is determined by RIA, but RIA estimates may not accurately reflect serum GH bioactivity. The available measures of GH bioactivity lack either sensitivity, specificity, or a physiologically relevant end point. The objective of this research was to develop a physiologically relevant GH bioassay which would not only measure the bioactivity of purified GH preparations, but would also have sufficient sensitivity to measure GH bioactivity in human serum. The method consisted of incubating murine 3T3-F442A adipocytes in serum-free medium containing BSA, 14C-glucose, and increasing concentrations of GH or test materials for 24 h, followed by measurement of conversion of glucose to lipid. Interference by nonspecific serum factors was reduced by the addition of 10 micrograms/liter insulin, 25 nM dexamethasone, and 37 nM estradiol to the medium. In the presence of 10 micrograms/liter insulin, 50 micrograms/liter insulin-like growth factor-1 did not alter the ability of GH to suppress lipid accumulation. Epinephrine and glucagon could suppress lipid accumulation but only at concentrations greatly in excess of the physiological range in serum. Twenty two thousand dalton hGH produced dose-dependent suppression of lipid accumulation which was linear between 0.625 and 10 micrograms/liter (r = 0.926; P = 0.0001) with a half-maximal response of 3.0 +/- 0.2 micrograms/liter (n = six experiments). The intra- and interassay coefficients of variation were 7% and 19%, respectively. The assay was specific for GH since addition of human PRL produced suppression of lipid accumulation only at concentrations where contamination of the preparation by GH became a significant factor. ACTH also suppressed lipid accumulation but only at doses of 1000 micrograms/liter or greater. Human placental lactogen and hLH, hFSH, and hTSH did not cross-react with GH in this assay. Addition of human serum did not alter the slope of ED50 of the GH dose-response curve. Pools of serum from prepubertal and pubertal boys and girls, subjects treated with arginine or insulin, a diabetic girl, and a boy with gigantism who had a serum GH content of 80 micrograms/liter by RIA and 40 micrograms/liter by bioassay, produced dose response curves parallel to that of the GH standard curve. Serum from patients with hypopituitarism did not produce significant suppression of lipid accumulation in any assay. Recovery of 5 micrograms/liter GH added to human serum was 94%. Twenty thousand dalton GH also suppressed lipid accumulation in this assay, but was 2-fold less potent than 22,000 dalton GH.(ABSTRACT TRUNCATED AT 400 WORDS)

Pituitary and recombinant deoxyribonucleic acid-derived human growth hormones alter glucose metabolism in 3T3 adipocytes.

3T3-F442A adipocytes were used to compare the effects on glucose metabolism of pituitary human (h) GH and methionyl-hGH produced by recombinant DNA techniques (met-hGH 22K). Pituitary hGH and met-hGH 22K were similar in their ability to inhibit glucose oxidation and lipid accumulation after 48 h. After 4 h of incubation, both forms of hGH stimulated glucose oxidation transiently in 3T3 adipocytes. A bacterially produced form of the 20,000-dalton variant of hGH (met-hGH 20K) also stimulated glucose oxidation at 4 h and inhibited glucose oxidation and lipid synthesis after 48 h. All three forms of hGH had a similar ability to inhibit [125I]iodo-met-hGH 22K binding to 3T3-adipocytes. Thus, met-hGH 22K and 20K directly produce in 3T3 adipocytes the transient stimulation and delayed inhibition of glucose metabolism attributed to pituitary hGH, indicating that these metabolic effects are intrinsic to the hGH molecule.

Acute effects of testosterone infusion and naloxone on luteinizing hormone secretion in normal men.

To evaluate the role of endogenous opioid pathways in the acute suppression of LH secretion by testosterone (T) infusion in men, we studied eight normal healthy volunteers who received a saline infusion, followed 1 week later by a T infusion (960 nmol/h) starting at 1000 h and lasting for 33 h. After 2 h of infusion (both saline and T), four iv boluses of saline were given hourly, and after 26 h of infusion, four hourly iv boluses of naloxone were given. Blood was obtained every 15 min for LH and every 30 min for T. T infusion increased the mean plasma T concentration 2.1-fold (18.7 +/- 2.1 to 39.5 +/- 3.5 nmol/L, saline vs. T infusion, P < 0.01). The mean plasma LH concentration was 7.9 +/- 0.5 IU/L during the saline control study and was decreased to 6.9 +/- 0.6 IU/L by the infusion of T (P < 0.05). LH pulse frequency was similar during both saline and T infusions (0.48 +/- 0.02 vs. 0.43 +/- 0.04 pulses/man.h, saline vs. T infusion). The mean LH pulse amplitude decreased from 4.3 +/- 0.4 IU/L during saline infusion to 3.3 +/- 0.2 IU/L during T infusion (P < 0.05). The administration of naloxone increased the mean plasma LH concentration significantly during saline infusion (7.6 +/- 0.4 to 10.0 +/- 0.9 IU/L, saline vs. naloxone boluses, P < 0.01), but not during T infusion (6.9 +/- 0.6 vs. 7.3 +/- 0.6 IU/L). LH pulse frequency increased significantly after the administration of naloxone during both saline and T infusions (0.54 +/- 0.04 to 0.71 +/- 0.08 pulses/man.h, saline vs. naloxone boluses during saline infusion, and 0.46 +/- 0.08 to 0.60 +/- 0.07 pulses/man.h during T infusion; P < 0.05). LH pulse amplitude was suppressed by T infusion, but administration of naloxone did not reverse this suppression. The mean amplitude of the LH response to exogenous GnRH (250 ng/kg) was decreased by T infusion from 48 +/- 13.5 to 31.2 +/- 8.5 IU/L (P < 0.01). Therefore, in men, the administration of naloxone increases LH pulse frequency during both saline and T infusions, but the acute suppression of LH pulse amplitude seen with T infusion was not reversed by naloxone. This pattern contrasts sharply with the effects of T infusion in pubertal boys, as elucidated by our earlier studies. The negative feedback effects of T on LH secretion are primarily hypothalamic in early pubertal boys and change to pituitary suppression in men.

Bioactive follicle-stimulating hormone responses to intravenous gonadotropin-releasing hormone in boys with idiopathic hypogonadotropic hypogonadism.

To test the hypothesis that exogenous pulsatile administration of GnRH will increase serum bioactive FSH (bFSH) levels, we studied four boys with suspected idiopathic hypogonadotropic hypogonadism (IHH). These boys presumably secreted relatively little GnRH. By virtue of their low baseline serum gonadotropin levels yet responsive pituitary gonadotrophs, these boys with IHH proved to be an excellent clinical model to test this hypothesis. Administration of GnRH (0.025 microgram/kg.dose) iv at 1- or 2-h intervals for 3-5 days resulted in an increase in serum bFSH after 91% of the GnRH doses. Serum immunoreactive FSH (iFSH) and LH (iLH) levels increased after 42% and 64% of the GnRH doses, respectively. Ninety percent of the iLH responses were concordant with bFSH responses, but only 33% of the iLH responses were concordant with iFSH responses. The serum bFSH responses occurred consistently within 20 min after GnRH administration and resulted in an increased serum bioactive to immunoreactive FSH ratio. By 60 min, serum bFSH levels had returned to preinjection levels. Serum testosterone and estradiol levels did not change during the period of GnRH administration in three of the four boys. We conclude that pulsatile, low dose iv GnRH administration in boys with IHH elicits significant serum bFSH increases by 20 min; the newly secreted FSH is preferentially enriched with increased in vitro FSH bioactivity, and it is rapidly cleared from serum (60 min). Therefore, serum bFSH measurements may provide a sensitive index of GnRH effects on the gonadotrophs.

Changes in serum immunoreactive and bioactive growth hormone concentrations in boys with advancing puberty and in response to a 20-hour estradiol infusion.

Acceleration of linear growth during puberty is associated with increased GH secretion, although the relationship between growth and GH is complex. As GH exists as a family of isoforms, some of which may not be identified by immunoassay, there may be alterations in isoform secretion during pubertal maturation that result in increased growth. The changes in serum immunoreactive and bioactive GH concentrations across pubertal maturation were determined in 30 boys, aged 6.5-19.3 yr, with idiopathic short stature or constitutional delay of adolescence. Data were grouped as follows: 1) 6 prepubertal boys with bone age 7 yr or less; 2) 5 prepubertal boys with bone age of more than 7 yr, 3) 10 boys in early puberty; 4) 9 boys with mid- to late puberty. Blood was obtained every 20 min from 2000-0800 h. An equal aliquot of each serum sample was pooled for determination of GH by bio- and immunoassays. The mean serum immunoreactive GH concentration increased from 2.1 +/- 0.3, 1.8 +/- 0.3, and 2.9 +/- 0.5 micrograms/L in groups 1, 2, and 3, respectively, to a peak of 4.6 +/- 0.7 micrograms/L in group 4 (P < 0.05 vs. groups 1-3). The mean serum GH bioactivity was 48 +/- 13 micrograms/L in group 1 and declined to 39 +/- 8 and 31 +/- 3 micrograms/L in groups 2 and 3, increasing to a maximum of 64 +/- 15 micrograms/L in group 4 (P < 0.05 vs. group 3). The ratio of bioactive to immunoreactive GH suggests that the biopotencies of secreted isoforms do not increase during pubertal maturation. The role of E2 in increasing GH secretion was characterized in 8 additional early pubertal boys. Each boy received a saline infusion from 1000-0800 h, followed 1 week later by an infusion of E2 at 4.6 nmol/m2.h. Blood was obtained every 15 min from 2200-0800 h for GH and LH and every 60 min for E2 and testosterone. An equal aliquot of each overnight serum sample was pooled for insulin-like growth factor I (IGF-I) and GH by immuno- and bioassays. The mean serum LH concentration decreased from 5.0 +/- 0.9 to 2.3 +/- 0.6 IU/L (P < 0.01), and the E2 concentration increased from 22 +/- 4 to 81 +/- 26 pmol/L (P < 0.01) during saline and E2 infusions, respectively. Mean serum GH concentrations as measured by immunoassay were similar during both infusions (6.6 +/- 1.4 vs. 9.7 +/- 2.1 micrograms/L; saline vs. E2 infusion, respectively). In contrast, the mean serum GH concentration, as measured by bioassay, decreased from 48 +/- 10 micrograms/L during saline infusion to 16 +/- 3 micrograms/L during E2 infusion (P < 0.05). The mean serum IGF-I concentration also decreased significantly from 116 +/- 17 to 93 +/- 15 micrograms/L (saline vs. E2 infusion, respectively; P < 0.05). Thus, although mean overnight serum GH concentrations increase in late puberty, whether measured by immuno- or bioassay, an acute increase in E2 produces an acute decline in serum GH bioactivity and a lesser decline in the serum IGF-I concentration. These unexpected changes indicate that E2 may affect pubertal growth and GH secretion in a complex or biphasic manner depending on the context in which it is administered.

Luteinizing hormone pulse characteristics in early pubertal boys are the same whether measured by radioimmuno- or immunofluorometric assay.

We tested the hypothesis that the improved sensitivity of immunofluorometric (IFMA) assays will lead to an increase in the number of detectable LH pulses compared to RIA in early pubertal boys, in whom LH secretion is low. To test this hypothesis we determined plasma LH concentrations in six pubertal boys (bone age, 12-16 yr) by IFMA and compared the results to RIA data reported previously. Each boy was given an infusion of saline, followed 1 week later by an infusion of testosterone (T; 960 nmol/h) for 33 h starting at 1000 h. Starting at 1200 h, blood was obtained every 15 min for LH determinations (RIA and IFMA) and every 30 min for T measurements. At the end of both studies, responses to GnRH (250 ng/kg) were assessed. The assay sensitivities for LH by RIA and IFMA (Delfia hLH Spec Pharmacia Diagnostics ENI, Columbia, MA) were 1.0 and 0.05 IU/L, respectively. LH pulses were identified by three independent pulse detection programs: Detect, Cluster, and Kushler-Brown. The correlation for LH values as measured by RIA and IFMA was highly significant (r = 0.81). There was a poor correlation between LH values determined by IFMA and RIA when LH values within 4 times the SD of each assay sensitivity were compared (r = 0.08; P = NS). T infusion suppressed LH pulse frequency by 40% and 66%, as determined by RIA and IFMA, respectively (P = NS). Using the Detect program, during the complete study in all 6 boys, 117 pulses of LH were identified by RIA and 93 by IFMA (79% ratio of detection IFMA/RIA). During saline infusion there were 73 vs. 69 LH pulses (94%), while during T infusion there were 24 vs. 44 LH pulses (55%), as detected by IFMA vs. RIA, respectively. Administration of naloxone did not accelerate LH pulse frequency during T infusion, as determined by either method. Changes in pituitary responses to exogenous GnRH also showed similar trends of augmentation by T infusion by both methods. We conclude that the use of IFMA does not lead to the anticipated increase in the detectability of LH pulsatility. Actually, fewer LH pulses were identified by IFMA in this group of boys. We speculate that this is due to the increased specificity of the IFMA assay. More significant was the finding that the physiological interpretation of the effects of T and naloxone on LH pulse frequency and responses to GnRH did not change whether LH was measured by RIA or IFMA.

Naloxone does not reverse the suppressive effects of testosterone infusion on luteinizing hormone secretion in pubertal boys.

In this study we wished to test whether, and if so when, the suppressive effects of testosterone on LH and, by inference, GnRH secretion are mediated via endogenous opioid pathways during male pubertal maturation. As a preliminary study, we evaluated the acute effects of a 24-h infusion of testosterone (T) in eight pubertal boys with constitutional delay of growth in order to determine the optimal time for administration of naloxone. Eight additional pubertal boys received a saline infusion, followed 1 week later by a similar T infusion starting at 1000 h and lasting for 33 h. After 2 h of infusion (both saline and T), four iv boluses of saline were given hourly, and after 26 h of infusion, four hourly iv boluses of naloxone were given. Blood was obtained every 15 min for LH and every 30 min for T measurements. T infusion increased the mean T concentration by 3.8-fold (P less than 0.001). Mean LH and LH pulse frequency were suppressed (P less than 0.01), and the sleep-associated increase in LH secretion was abolished. Naloxone administration during the infusion of T did not reverse the suppression of LH secretion. Compared to the saline control period, mean LH was significantly lower during T infusion during the time naloxone boluses were given (4.5 +/- 0.9 vs. 5.9 +/- 1.1 IU/L, T infusion and naloxone boluses vs. saline respectively, P less than 0.01). Although the suppression of LH pulse frequency remained significantly lower than that during the saline control period (0.23 +/- 0.04 pulses/boy.h during T infusion and saline boluses; 0.33 +/- 0.04 pulses/boy.h during T infusion plus naloxone boluses; 0.44 +/- 0.06 pulses/boy.h during saline infusion and saline boluses). Naloxone increased mean LH and LH pulse frequency only in the four older, more mature boys during the infusion of saline. Pituitary responsiveness to exogenous GnRH was not altered by infusion of T. We conclude that acute administration of T suppresses LH secretion and, by inference, GnRH secretion at all stages of pubertal maturation in boys. These negative feedback effects, however, cannot be reversed by coadministration of naloxone, even in mid- to late pubertal boys who respond to naloxone with increased pulsatile secretion of LH. These studies suggest that during pubertal maturation in boys, endogenous opioid pathways do not play a major role in the regulation of the negative feedback effects of T.

Increased luteinizing hormone pulse frequency during sleep in early to midpubertal boys: effects of testosterone infusion.

Gonadotropin secretion is pulsatile in prepubertal and early pubertal boys, and the onset of puberty is characterized by a sleep-associated rise in LH pulse amplitude. To determine whether an augmentation in LH pulse frequency as well as amplitude occurs at the onset of puberty, we studied gonadotropin secretion in 21 early to midpubertal boys. Blood samples were taken every 20 min (every 15 min in 4 boys) for LH determinations. A 2-fold increase in LH pulse frequency occurred during the nighttime sampling period (2200-0400 h) compared to that in the hours when the boys were awake (1000-2200 h). The maximum frequency (0.7 pulses/h) occurred between 2400 and 0200 h. The mean plasma LH concentration increased during the night from 2.3 +/- 0.2 (+/- SE) mIU/mL (2.3 +/- 0.2 IU/L) between 2000-2200 h to a maximum of 6.2 +/- 0.4 (6.2 +/- 0.4 IU/L) between 0200-0400 h. The mean plasma LH decreased to 5.5 +/- 0.4 mIU/mL (5.5 +/- 0.4 IU/L) between 0400-0600 h and to 4.2 +/- 0.5 (4.2 +/- 0.5 IU/L) between 0600-0800 h. Plasma testosterone rose during the night to a mean maximum value of 2.4 +/- 0.5 (+/- SE) ng/mL (8.3 +/- 1.7 nmol/L). This finding suggested that the rise in testosterone might play a role in decreasing LH secretion during the later hours of sleep (after 0400 h). To address this question and to study further the effects of testosterone in early puberty, we measured plasma LH concentrations every 10 min from 2000-0800 h in 8 early to mid-pubertal boys before and during short term testosterone administration. Saline or testosterone at a concentration of 9.33 micrograms/mL (32 mumol/L) was infused at a rate of 10 mL/h from 2100-1200 h to shift the nighttime testosterone rise 3 h earlier than would occur spontaneously. Blood samples were obtained every 10 min for LH and every 30 min for testosterone determinations from 2000-0800 h. Pituitary responsiveness was assessed by administering sequential doses of synthetic GnRH (25 and 250 ng/kg) at 1000 and 1200 h, respectively. The nighttime increase in LH pulse frequency and mean plasma LH concentration occurred between 2300 and 0200 h despite testosterone infusion. However, testosterone infusion was associated with significantly lower mean plasma LH concentrations from 0200-0800 h compared to those on the night of the saline infusion. Pituitary responsiveness to synthetic GnRH was unaltered by testosterone administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with glucocorticoid resistance and a glucocorticoid-resistant New World primate species.

Members of a previously reported family with glucocorticoid resistance and several New World primates have high plasma cortisol concentrations without any signs of glucocorticoid excess. The glucocorticoid receptor in circulating leukocytes and cultured skin fibroblasts from these patients and the animals is characterized by a decreased affinity for dexamethasone. On the other hand, the cell content of receptor is similar to that of corresponding tissues of normal humans. Detailed biochemical-biophysical studies of the glucocorticoid receptor in this familial syndrome and animal model became possible with the use of Epstein-Barr virus-transformed lymphocyte lines. Cell lines from patients with this syndrome and from the marmoset (Saguinus oedipus) contained decreased amounts of glucocorticoid receptors with concomitant decreases in nuclear receptor content compared to cultured Epstein-Barr virus-transformed lymphocytes from normal human subjects. This may reflect diminished induction of glucocorticoid receptor during viral transformation of cells from the patients and the animal model. Receptors from a severely affected glucocorticoid-resistant patient and the marmoset had decreased affinity for dexamethasone. Evidence for a mild affinity defect of the glucocorticoid receptor in a patient with asymptomatic glucocorticoid resistance was obtained by increased hormone-receptor dissociation at an elevated temperature. Thermal stability, mero-receptor formation, thermal activation of cytosolic receptor, and mol wt of receptors from all cell lines were normal. Only the receptors of the severely affected patient had a discernible defect in temperature-induced activation of intact cells. We conclude that the major detectable change in the receptor in both the patients and the animal model is the decreased affinity for glucocorticoid. Viral receptor induction is decreased in both patient and marmoset cells. The physiological relevance of this phenomenon is not known. Gross receptor molecule changes or changes in its stability at higher temperatures were not found. Mixing studies did not show involvement of cytosolic modifiers or inhibitors. Mutation(s) of the receptor molecule leading to low affinity for the hormone is the most likely explanation of the isolated glucocorticoid resistance in the patients. The glucocorticoid resistance of the New World primate, which is part of generalized steroid hormone resistance, appears to be a result of more complex changes.

Comparison of the neuroendocrine control of pubertal maturation in girls and boys with spontaneous puberty and in hypogonadal girls.

Puberty in boys is characterized by a nocturnal increase in mean LH concentration and LH pulse frequency. To determine whether similar mechanisms exist in girls, nocturnal serum LH concentrations were determined in 16 girls with constitutional delay of adolescence or idiopathic short stature who had or have subsequently been shown to have spontaneous puberty. Mean LH and LH pulse frequency and amplitude were analyzed in 3-h blocks and compared to those in 20 pubertal boys. Girls had an increase in mean LH concentration from 3.6 +/- 0.7 IU/L at 2000-2250 h to 4.8 +/- 0.9 IU/L at 0200-0450 h. LH pulse frequency increased from 0.27 +/- 0.11 pulses/girl.h at 2000-2250 h to 0.54 +/- 0.10 pulses/girl.h at 0200-0450 h. The increase in LH pulse amplitude, from 2.0 +/- 0.8 IU/L at 2000-2250 h to 4.1 +/- 1.1 IU/L at 2300-0150 h, did not achieve statistical significance because many girls had no pulses from 2000-2250 h. With advancing age, the day/night differences in LH concentration and LH pulse frequency disappeared in girls, but were preserved in boys of same pubertal stage. The effect of lack of estrogen on LH pulse characteristics was inferred by analyzing the LH profiles of 15 girls with gonadal dysgenesis who were age-matched to girls with spontaneous puberty. The girls with gonadal dysgenesis had an increase in mean LH concentration after 0200 h, but LH pulse frequency was rapid in all time blocks; the nocturnal increase in LH concentration was secondary to a significant increase in LH pulse amplitude. Older girls with gonadal dysgenesis had a loss of nighttime augmentation of LH secretion similar to that seen in girls with spontaneous puberty. These data suggest that the apparent slower LH pulse frequency encountered in girls with spontaneous puberty during waking hours may be related to estrogen suppression of LH pulse amplitude, which masks the true daytime LH pulse frequency. With or without pubertal estrogen exposure, developmental progression of LH secretion occurs more rapidly in girls than in boys. Thus, intrinsic sex differences exist in the timing and tempo of endocrine control of pubertal maturation between boys and girls.

Nocturnal naloxone fails to reverse the suppressive effects of testosterone infusion on luteinizing hormone secretion in pubertal boys.

LH secretion is maximal during the night in pubertal boys, and testosterone (T) administration blunts this nocturnal rise of LH. We have previously shown that in pubertal boys, the acute negative feedback effects of T infusion on LH secretion during the daytime cannot be reversed by opioid receptor blockade. To determine whether the nocturnal secretion of LH in early puberty is regulated by endogenous opioid pathways, we determined whether naloxone during the night affected LH secretion or T-mediated suppression of LH secretion. Seven pubertal boys (bone age, 11-13.5 yr) were given a control infusion of saline, followed 1 week later by an infusion of T at 960 nmol/m2.h for 41 h starting at 2000 h. During both saline and T infusions, six iv boluses of saline were given hourly beginning at 2400 h on the first day, and six iv boluses of naloxone (0.1 mg/kg each) were given hourly beginning at 2400 h on the second day. Starting at 2200 h, blood was obtained every 15 min for LH and every 30 min for T determinations for 14 h each night. Pituitary responsiveness was assessed at the end of each study night by i.v. bolus administration of 250 ng/kg synthetic GnRH. T infusion increased the mean T concentration 6-fold (P < 0.0001) and suppressed the mean plasma LH concentrations from 5.6 +/- 0.6 to 3.8 +/- 0.6 IU/L (P < 0.01). The nocturnal augmentation of LH secretion was suppressed by the infusion of T, and this suppression was not reversed by naloxone. The mean nighttime plasma LH (2400-0600 h) was 8.1 +/- 1.1 IU/L during the control saline infusion and 5.1 +/- 0.6 IU/L during the T infusion (P < 0.01). The mean LH level was 4.0 +/- 0.7 IU/L during the administration of naloxone boluses concomitantly with the T infusion, not significantly different from that during the T infusion. Likewise, LH pulse frequency during the same time period was decreased by T infusion from 0.6 +/- 0.1 to 0.36 +/- 0.04 pulses/boy.h (P < 0.05), and it was unaltered by coadministration of naloxone (0.38 +/- 0.12 pulses/boy.h). Naloxone administration during the saline infusion did not increase either the mean plasma LH concentration (7.5 +/- 0.7 IU/L; P = NS vs. saline control) or the LH pulse frequency (0.69 +/- 0.1 pulses/boy.h; P = NS vs. saline control). Pituitary responsiveness to GnRH was similar on each of the 4 nights during either saline or T infusions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute effects of estradiol infusion and naloxone on luteinizing hormone secretion in pubertal boys.

We have shown previously in pubertal boys that testosterone (T) suppresses the nocturnal augmentation of luteinizing hormone (LH) secretion principally by decreasing LH pulse frequency. As T can be aromatised to estradiol (E2), and E2 effects on LH secretory dynamics may be separate from those of T, we examined the effects of acute E2 infusion on LH secretion in pubertal boys. Opioid receptor blockade has been reported to increase LH secretion after estradiol suppression in adult men, so we also examined whether naloxone might augment LH secretion during E2 treatment in pubertal boys. Starting at 1000 h, eight pubertal boys were given a 33 h saline infusion, followed 1 week later by an E2 infusion at 4.6 nmol/m2/h. During both infusions, four iv boluses of saline were given hourly beginning at 1200 h on the first day, and four naloxone iv boluses, 0.1 mg/kg each, were given hourly beginning at 1200 h on the second day. Blood was obtained every 15 min for LH, and every 60 min for T and E2, from 1200 h until the end of the infusion. Pituitary responsiveness to gonadotropin-releasing hormone (GnRH) was assessed after both infusions by iv administration of 250 ng/kg synthetic GnRH. Estradiol infusion increased the mean plasma E2 concentration from 23 +/- 4 to 46 +/- 6 pmol/L (P < 0.01) and suppressed mean plasma T from 4.9 +/- 1.4 to 3.0 +/- 3.5 nmol/L (saline vs. E2 infusion, P < 0.05). The overall mean LH was suppressed by E2 infusion from 3.7 +/- 0.5 to 2.2 +/- 0.4 IU/L (saline vs. E2 infusion, P < 0.01). LH pulse frequency was suppressed by 50%, whereas mean LH pulse amplitude was not different between saline and E2 infusions. Administration of naloxone did not alter the mean LH, LH pulse frequency, or amplitude during either saline or E2 infusions. Pituitary responsiveness to exogenous GnRH was similar during both infusions. These studies indicate that E2 produces its negative feedback in pubertal boys principally by suppression of LH pulse frequency, and naloxone does not reverse these suppressive effects. Thus E2 suppression of LH secretion is mediated by a decrease of hypothalamic GnRH secretion that is independent of endogenous opioid pathways.

Absence of pubertal gonadotropin secretion in girls with McCune-Albright syndrome.

Precocious puberty in girls with McCune-Albright syndrome has been attributed in some cases to early activation of the hypothalamic-pituitary-gonadal axis and in other cases to sex steroid secretion by apparently autonomous ovarian cysts. We evaluated serum gonadotropins and sex steroids in six girls (aged 1-9 yr) with McCune-Albright syndrome. The children had Tanner stage II-IV pubertal development. In five patients, nocturnal gonadotropin concentrations and the gonadotropin response to LHRH were within the normal range for prepubertal children. Thus, the precocious puberty in these patients could not be explained by activation of the hypothalamic-pituitary-ovarian axis. One child had high amplitude nocturnal pulses of serum LH and a LH-predominant response to LHRH. She was the oldest of the six girls and had a bone age of 13.5 yr which is within the range in which hypothalamic-pituitary-ovarian activation normally occurs. The children all had ovarian enlargement and ovarian cysts determined by ultrasound. It appears that precocious puberty in McCune-Albright syndrome may result from ovarian estrogen secretion in the absence of normal pubertal activation of the hypothalamic-pituitary-ovarian axis.

In pubertal girls, naloxone fails to reverse the suppression of luteinizing hormone secretion by estradiol.

Estradiol (E2) negative feedback on LH secretion was examined in 10 pubertal girls, testing the hypothesis that E2 suppresses LH pulse frequency and amplitude through opioid pathways. At 1000 h, a 32-h saline infusion was given, followed 1 week later by an E2 infusion at 13.8 nmol/m2 x h. During both infusions, four iv boluses of saline were given hourly beginning at 1200 h, and four naloxone iv boluses (0.1 mg/kg each) were given hourly beginning at 1200 h on the following day. Blood was obtained every 15 min for LH determination and every 60 min for E2 determination from 1200 h to the end of the infusion. E2 infusion increased the mean serum E2 concentration from 44+/-17 to 112+/-26 pmol/L (P < 0.01). The mean LH concentration between 2200-1200 h decreased from 3.19+/-0.89 to 1.99+/-0.65 IU/L (P = 0.014), and LH pulse amplitude decreased from 3.4+/-0.6 to 2.6+/-0.5 IU/L (P = 0.0076). Although there were 1.2 fewer pulses during E2 infusion compared to saline infusion, differences did not reach significance (P = 0.1; 95% confidence interval for the difference, -3.5, 1.1). Pituitary responsiveness to GnRH, assessed at the end of the infusion by administering 250 ng/kg GnRH iv, did not change during E2 infusion. The effect of naloxone blockade of opioid activity on LH secretion was determined by assessing the area under the curve (AUC) from 1200-1600 h. During saline infusion, the LH AUC was 1122+/-375 IU/L during saline boluses and 1575+/-403 IU/L during naloxone boluses (P = 0.39). When E2 was infused, the LH AUCs during saline and naloxone boluses were 865+/-249 and 866+/-250 IU/L, respectively. Thus, in pubertal girls: 1) E2 decreases the LH concentration and LH pulse amplitude; 2) the main site of negative feedback effect of E2 appears to be at the level of the hypothalamus; 3) an increase in LH secretion after naloxone administration could not be demonstrated in these girls and may depend on the maturity of the hypothalamic-pituitary-gonadal axis; and 4) opioid receptor blockade does not reverse the E2 inhibition of LH secretion even in the most mature girls. Thus, E2 suppression of LH secretion in pubertal girls appears to be mediated by a decrease in hypothalamic GnRH secretion that is independent of opioid pathways.

Differential regulation of serum immunoreactive luteinizing hormone and bioactive follicle-stimulating hormone by testosterone in early pubertal boys.

The microheterogeneity and bioassayable activity of serum FSH (B-FSH) can be regulated by exogenous GnRH in boys with idiopathic hypogonadotropic hypogonadism and by estrogen in a women with gonadal dysgenesis, presumably via hormonally mediated changes in the degree of FSH glycosylation. To test the hypothesis that testosterone (T) regulates the circulating forms of B-FSH, we raised the serum T levels of early pubertal boys to adult levels. In this model, high dose T inhibits the pubertal nocturnal augmentation of LH secretion, apparently through decreased GnRH secretion. This model allowed us to test a second hypothesis, that B-FSH is a sensitive indicator of hypothalamic GnRH release. The boys were studied on two consecutive weekends, during which they received either saline (S) or T infusions. Beginning at noon on the study day, after an overnight acclimatization, the boys received either S or T at 33% or 100% of the adult male production rate. Blood was sampled from 2000-0800 at 10-min intervals for immunoactive LH and FSH (I-FSH) and for B-FSH, as determined by the in vitro Sertoli cell aromatase induction assay, and at 30-min intervals for T. Gonadotropin levels were analyzed as mean hourly or 3-h concentrations and as pulse profiles by two established objective peak detection programs, Cluster and Detect. During S treatment, mean LH increased after the onset of sleep (P = 0.0006) and, after plateauing for several hours, declined to baseline in the early morning hours. Mean levels of B-FSH were also minimally (but significantly) increased after the onset of sleep (P = 0.046) and paralleled the decline noted for LH. Mean levels of I-FSH did not demonstrate a diurnal rhythm. The effect of T was gonadotropin specific. High dose T abolished the nocturnal elevation in mean LH concentrations, but had no effect on the nocturnal elevation of B-FSH (P less than 0.05) or on I-FSH levels. The LH pulse frequency was greatest from 2300-0450 h, during S treatment (P = 0.016). The pulse frequency of B-FSH was also minimally increased after the onset of sleep (P = 0.045). The T infusion abolished the nocturnal increase in LH pulse frequency, without an effect on B-FSH pulse frequency. B-FSH pulse frequency exceeded LH pulse frequency during S treatment (8.0 +/- 0.7 pulses/12 h vs. 5.5 +/- 0.4), and B-FSH pulses persisted throughout the night. The pulse amplitudes of LH and B-FSH were not affected by T.(ABSTRACT TRUNCATED AT 400 WORDS)

Testosterone infusion reduces nocturnal luteinizing hormone pulse frequency in pubertal boys.

Administration of testosterone (T) can inhibit LH secretion in early pubertal boys. However, the GnRH pulse generator is relatively resistant to the effects of T, since T infusion beginning at 2100 h, 3 h before the usual nighttime increase in T, does not suppress the characteristic increase in LH pulse frequency or amplitude associated with the onset of sleep in early pubertal boys. To test the hypothesis that the hypothalamic-pituitary axis must be exposed to T for a longer duration to suppress the nocturnal rise in LH pulse frequency and amplitude, we infused saline or T at one third the adult male production rate (320 nmol/h), beginning at 1200 h on two consecutive weekends in each of eight early to midpubertal boys. Blood was obtained from 2000-0800 h every 10 min for LH and every 30 min for T measurements. T infusion increased the mean plasma T concentration from 6.9 +/- 1.7 to 11.8 +/- 1.4 nmol/L (P less than 0.01) between 2000-0800 h. Despite the T infusion, the nocturnal rise in mean LH concentration and LH pulse frequency persisted, suggesting that the nocturnal amplification of LH, and by inference GnRH, secretion is resistant to the negative feedback effects of T. A higher dose of T, approximating the adult male production rate (960 nmol/h), was given to eight additional boys beginning at 1200 h. The mean T concentration increased from 4.2 +/- 1.7 to 20.8 +/- 3.1 (P less than 0.001) nmol/L between 2000-0800 h. The mean plasma LH concentration was suppressed by T infusion from 5.2 +/- 0.5 to 2.9 +/- 0.4 IU/L, and LH pulse frequency decreased from 0.50 +/- 0.04 to 0.27 +/- 0.11 pulses/boy/h (P less than 0.01). There was no nocturnal amplification of LH secretion, but high amplitude LH pulses did occur during the night in six of the eight boys. The low dose T infusion had no effect on pituitary LH release by exogenous GnRH. With the high dose T infusion, however, the ability of GnRH, at 25 ng/kg but not at 250 ng/kg, to release pituitary LH was amplified. Thus, T supplementation at one third the adult male production rate does not blunt the sleep-associated nighttime rise in LH pulse frequency or LH concentration. T infusion approximating the adult male production rate suppresses the nocturnal increase in LH pulse frequency and mean LH concentration, and high amplitude, slow frequency LH pulses similar to patterns seen in adult men persist.(ABSTRACT TRUNCATED AT 400 WORDS)

Variable response to a long-acting agonist of luteinizing hormone-releasing hormone in girls with McCune-Albright syndrome.

Six girls with McCune-Albright syndrome were treated for at least 2 months with the long-acting LHRH agonist D-Trp6-Pro9-NEt-LHRH, which previously was found to be an effective treatment for true precocious puberty. Nocturnal and LHRH-stimulated serum gonadotropin levels and plasma estradiol levels were measured before treatment and after 2-3 months of treatment. Five of the six girls had no decrease in serum gonadotropin or plasma estradiol levels during therapy, and their pubertal signs were unaffected by treatment. All five of these girls had serum gonadotropin levels that were within or below the normal prepubertal range. The sixth girl, who had gonadotropin levels in the normal pubertal range before treatment, had decreased serum gonadotropin and plasma estradiol levels during 1 yr of LHRH analog therapy. This was associated with cessation of menses and regression of secondary sexual changes. The failure of LHRH analog to modify the course of precocious puberty in the five patients with prepubertal serum gonadotropin concentrations is further evidence that the mechanism of precocious puberty in most girls with McCune-Albright syndrome differs from that in patients with true precocious puberty.