Pulsatile administration of gonadotropin-releasing hormone does not alter the follicle-stimulating hormone (FSH) isoform distribution pattern of pituitary or circulating FSH in nutritionally growth-restricted ovariectomized lambs.

The experimental induction of puberty by GnRH administration to prepubertal lambs increases serum bioactive FSH (B-FSH) as measured in the rat Sertoli cell aromatase induction bioassay. Serum immunoreactive FSH (I-FSH) levels are unchanged. The increase in serum B-FSH is associated with an increase in the proportion of less acidic and more biopotent FSH serum isoforms. However, it is unknown if this effect of GnRH on serum FSH microheterogeneity is direct or mediated by gonadal factors. We have used the nutritionally growth-restricted ovariectomized lamb as a model of the neuroendocrine regulation of FSH isoform microheterogeneity. With this model, the hypothalamic-pituitary component of the neuroendocrine axis may be isolated from gonadal factors. In the present study, using the nutritionally growth-restricted ovariectomized lamb as a model, we investigated the role of GnRH on the regulation of FSH microheterogeneity. Specifically, we tested the hypothesis that GnRH increases the proportion of the less acidic (more biopotent) serum FSH isoforms. As an in vitro correlate, we investigated the effect of GnRH on gonadotropin secretion and FSH isoform distribution in ovine pituitary explant cultures. Seven ovariectomized nutritionally restricted lambs were administered GnRH (i.v., 2 ng/kg) for 36 h (at 2-h intervals for 24 h, then hourly for the final 12 h). Six others served as controls. Blood samples were withdrawn at 12-min intervals during the last 4 h for the measurement of serum immunoactive LH (I-LH) and I-FSH. Pituitary homogenates and serum from four animals from each group were individually chromatofocused, and the FSH isoform distribution patterns were determined. Pulsatile administration of GnRH to nutritionally growth-restricted lambs increased circulating I-LH concentrations from 0.6 +/- 1.0 to 5.9 +/- 3.1 ng/ml (P < 0.01), but did not significantly change circulating I-FSH (4.9 +/- 1.8 vs. 11.5 +/- 4.2 ng/ml) nor B-FSH concentrations (3.9 +/- 1.2 vs. 5.7 +/- 1.5 ng/ml). The pituitary content of I-FSH, B-FSH, and I-LH were unchanged. Neither serum nor pituitary FSH isoform distribution patterns were altered by pulsatile GnRH administration. However, compared to the pituitary FSH isoforms, a higher percentage of circulating FSH isoforms eluted in the salt peak of both groups of lambs. Similar to the in vivo studies, in vitro, GnRH increased the release of I-LH, as well as I-FSH, from pituitary explants, but did not significantly change the FSH isoform distribution in either the pituitary explant or media.(ABSTRACT TRUNCATED AT 400 WORDS)

Follicle-stimulating hormone signal transduction: role of carbohydrate in aromatase induction in immature rat Sertoli cells.

Receptor activated adenylate cyclase acts as a major transmembrane signalling system. It is widely accepted that upon binding to its receptor, follicle-stimulating hormone (FSH) activates the cAMP-dependent pathway which in turn mediates FSH-induced estradiol production in Sertoli cells. Studies utilizing several chemically derived variants of FSH have demonstrated that these variants bind to the FSH receptors with equal avidity but differ in their ability to activate cAMP-dependent pathways. Since cAMP is believed to be the second messenger responsible for FSH signal transduction, we tested two hypotheses: (1) that the effects of different oFSH variants on cAMP production and aromatase induction (as measured by estradiol production) would be in parallel; and (2) that deglycosylated ovine FSH (DG-oFSH) would antagonize the ability of intact oFSH to stimulate aromatase induction, similar to its reported antagonistic effect on cAMP production. Immature rat (7- to 10-day-old) Sertoli cells were cultured and the effects of several different oFSH variants on cAMP production and/or aromatase induction were tested. The variants tested were native oFSH, DG-oFSH, asialo oFSH (AS-oFSH), a recombinant of intact LH alpha and FSH beta (alpha + beta) and a recombinant of deglycosylated LH alpha and intact FSH beta (DG alpha + beta). Both native oFSH and alpha + beta recombinant at relatively large doses (10 ng) elicited a significant increase in extracellular cAMP accumulation as well as total cAMP production. In contrast, DG-oFSH did not produce an increase in cAMP even at 10-fold higher doses than native oFSH. Intracellular cAMP concentrations did not increase following stimulation with native oFSH, DG-oFSH or DG alpha + beta. In contrast to the divergent effects of oFSH and DG-oFSH on cAMP production all variants of oFSH stimulated estradiol production from Sertoli cells albeit with varying potencies. The sensitivity (minimal effective dose) and ED50 (dose at which half maximal response is achieved) of the estradiol (E2) response curve to increasing concentrations of native oFSH were 0.025 +/- 0.01 and 0.33 +/- 0.05 ng, respectively. Asialo-oFSH (AS-oFSH) increased E2 production with a potency (comparative dose required for effect) similar to that of native oFSH.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

Nocturnal serum growth hormone concentration is not augmented by short-term testosterone infusion in pubertal boys.

Chronic exposure to testosterone (T) increases growth hormone (GH) secretion. To determine whether acute exposure to T would also enhance GH secretion, we infused saline, followed 1 wk later by T, for 18-24 h at one-third the adult male production rate in 12 pubertal boys and at the adult male production rate in eight additional pubertal boys. Blood was obtained every 20 min for GH and every 30 min for T from 2000-0800 h. Though infusion significantly increased serum T concentrations in all 20 boys, mean GH concentration, GH pulse frequency, and GH pulse amplitude did not increase compared to the saline infusion night. The secretory dynamics of GH as a function of 3-h time blocks from 2000-0800 h were also determined in the eight boys who received the higher dose of T. The profile for mean GH concentration, pulse frequency, pulse amplitude, and peak area were not affected by acute infusion of T at concentrations sufficient to alter LH secretion. This suggests that, at least in pubertal boys, one must be exposed to T for a period longer than 12-18 h to induce increased GH secretion.

Induction of intestinal microsomal enzymes by polycyclic aromatic hydrocarbons.

1. The efficacy of induction of intestinal microsomal aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin o-deethylase and cytochrome P-450 by various polycyclic aromatic hydrocarbons were studied. 2. The greatest induction of the specific MFO activities tested was produced by 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) with 3-methyl-cholanthrene (MC) a close second. 3. Rank order of efficacy of induction of spectrally determined cytochrome P-450 was 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), Arochlor 1254 (ARO), 3-methylcholanthrene (MC) and benz(a)anthracene(BA). 4. No unique protein peaks were found when the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MC treated intestinal microsomes were prepared.

Influence of protein and RNA synthesis inhibitors on methylcholanthrene-mediated induction of aryl hydrocarbon hydroxylase activity in intestinal mucosa.

The sensitivity of the 3-methylcholanthrene (MC)-mediated induction of aryl hydrocarbon hydroxylase (AHH) activity to pretreatment with inhibitors of protein and RNA synthesis was studied. Cordycepin significantly inhibited the induction at 12 h but not at 3 h post-treatment with MC. Both cycloheximide and actinomycin D significantly inhibited the MC-mediated induction at 3 and 12 h after MC treatment. An MC-mediated stimulation of 3H-leucine incorporation into microsomal protein in vivo was observed at 12 h following MC treatment. A cell-free protein synthesis system employing intestinal ribosomes was developed. The rate of protein synthesis (3H-leucine incorporation/mg rRNA/3 min) and the number of active ribosomes (3H-peptidyl-puromycin formed/mg rRNA) were determined. No effect of in vivo MC treatment was observed in subsequently isolated intestinal ribosomes at either 3, 9 or 18 h post-treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on intestinal microsomes prepared from control, corn-oil- and MC-treated rats 18 h after treatment. The microsomal proteins depicted only slight quantitative changes in microsomal proteins following MC treatment, and no unique protein peaks. These results may explain the lack of detectable stimulation of the activity of isolated ribosomes. In addition, the methods used may lack sufficient sensitivity to detect the small net change in protein necessary to account for the increase in AHH activity seen after MC induction.

Endogenous dopaminergic dysfunction: a novel form of human growth hormone deficiency and short stature.

The purpose of this study was to determine if combined therapy with dopaminergic drugs (DA), i.e. L-dopa or bromocriptine, and exogenous human GH (hGH) could increase growth velocity in hypopituitary children. Twelve prepubertal hypopituitary children (eight boys and four girls; bone age, 1.5-9.5 yr), divided into two groups, each received hGH alone, DA alone, and DA and hGH. Group I (n = 6) received L-dopa (15 mg/kg, orally) at 6-h-intervals during DA and combined DA and hGH therapy. Group II (n = 6) received bromocriptine (1.25 mg, orally) every 12 h during DA and combined DA and hGH therapy. Both groups were given hGH (0.1 IU/kg) three times per week during hGH and combined hGH and respective DA treatment. The study included three 6-month treatment periods of DA, hGH, and combined DA and hGH therapy. The mean growth rates (centimeters per 6 months, +/- SD) before treatment and during the three study periods for group I were 1.7 +/- 0.2, 3.3 +/- 0.8, 3.4 +/- 0.4, and 3.9 +/- 0.7, respectively. Group II results were 1.4 +/- 0.3, 2.3 +/- 0.8, 5 +/- 1.6, and 3.7 +/- 1.1. Mean and peak hGH concentrations, measured every 30 min for 9 h at the end of each study period, increased significantly in five patients, from 15 +/- 3 (+/- SE) ng/ml during hGH therapy to 30 +/- 5 ng/ml during DA and hGH treatment. The mean peak hGH values rose from 24 +/- 4 to 45 +/- 5 (+/- SE) ng/ml. In conclusion, addition of dopaminergic agents to hGH therapy potentiates growth in some hypopituitary children. The increased growth and hGH responses to L-dopa or bromocriptine suggest impaired endogenous GH release. Dopaminergic therapy alone or in combination with exogenous hGH may be efficacious in some hypopituitary children.

Evidence for dopaminergic stimulation of growth velocity in some hypopituitary children.

The purpose of this study was to determine if endogenous hGH release, hence growth, in hypopituitary children could be potentiated by therapy with dopaminergic (DA) drugs namely, L-dopa or bromocriptine. The effect of DA therapy on other endocrine function was also examined. Subjects were nine prepubertal children (four girls and five boys) with bone ages (BA) ranging from 1.5-9.5 yr. They were diagnosed as having idiopathic GH deficiency on the basis of: 1) failure to grow at normal rates 2) lack of GH response to two provocative stimuli (oral L-dopa, and insulin-induced hypoglycemia) and 3) low somatomedin-C concentrations for sex and age. They were divided into two groups. Group I (n = 4) received L-dopa (15 mg/kg, orally, every 6 h) for 6 months. Group II (n = 5) received bromocriptine (1.25 mg, orally, every 12 h) for 6 months. At the end of 6 months of DA therapy, both groups received human GH (hGH) im (0.1 IU/kg, thrice weekly) for 6 months. The growth rate in group I increased to 5.7 +/- 0.6 (+/- SE) cm/yr during the 6 months from a pretreatment rate of 3.4 +/- 0.2 cm/yr. Individual increments ranged from 30-94% above pretreatment growth rates. Three of the four children had significantly increased height increments, and two children achieved growth rates normal for their BA. Similarly, the growth rate in group II increased to 4.8 +/- 0.8 cm/yr from the pretreatment rate of 2.9 +/- 0.3 cm/yr. Individual increments ranged from 46-100% above pretreatment growth rates. Three children in group II had significantly increased height increments, and two children had normal growth rates for BA. The growth increments during L-dopa therapy occurred in the three children who had significant increases in hGH and somatomedin-C; of the three children with significant growth increments during bromocriptine therapy, two had increases in somatomedin-C, and one achieved a normal peak hGH value. hGH therapy caused further acceleration of growth velocities in the majority of patients. DA therapy had no significant effect on basal gonadotropin, gonadal steroids, T4, TSH, or morning cortisol concentrations in the majority of children compared with their pretreatment values. The following conclusions were reached. Dopaminergic therapy by itself, i.e. L-dopa or bromocriptine administration, induced linear growth in some hypopituitary children without significantly affecting basal concentrations of LH, FSH, gonadal steroids, T4, TSH, or cortisol. The effect this therapy could have in potentiating exogenous GH and/or possible GRH therapy is worthy of further investigation.

Hepatic glutathione levels in D-penicillamine-fed ethanol-dependent rats.

Ethanol oxidation is accomplished primarily by alcohol dehydrogenase. However, a microsomal system involving hydrogen peroxide formation operates at elevated ethanol concentrations. Removal of the resultant hydrogen peroxide may depend on the activity of glutathione peroxidase. In the study, we have examined the effect of chronic ethanol exposure on hepatic glutatione levels and found that ethanol exposure resulted in elevations of hepatic reduced and oxidized glutathione. The dietary inclusion of the sulfhydryl amino acid, D-penicillamine, increased hepatic reduced glutathione (GSH) in both ethanol-dependent and control rats. However, D-penicillamine did not have a differential effect on hepatic GSH when comparing ethanol-dependent and control animals. Following two weeks exposure, the exclusion of ethanol and/or D-penicillamine from the diet for 24 hours resulted in a significant decrease in hepatic GSH.

Acetaminophen-induced glutathione depletion in diabetic rats.

The time course of acetaminophen-induced hepatic glutathione depletion and recovery in control and streptozotocin-diabetic female rats was investigated. Initial hepatic glutathione levels were significantly higher in control animals. After a 1 g/kg intraperitoneal dose of acetaminophen, maximum depletion of reduced glutathione (GSH) occurred within 2 h in diabetics and began to increase thereafter, while maximum GSH depletion occurred at approx. 4 h in controls and did not begin to return to normal until after 12 h. The time course for changes in hepatic oxidized glutathione (GSSG) after acetaminophen administration followed that for GSH in both control and diabetic rats. Diurnal variations in hepatic glutathione levels do occur, but these variations can not explain the changes which occur following acetaminophen administration or the differences observed between control and diabetic rats. Diabetic rats are more susceptible to the glutathione depleting effects of high-dose acetaminophen, suggesting that the diabetic may be less able to detoxify drugs and foreign chemicals.

Benzo[a]pyrene metabolism by hepatic and extrahepatic tissues in streptozotocin-diabetic rats.

The metabolism of benzo[a]pyrene (BP) by microsomes from hepatic and extrahepatic tissues of female diabetic rats was investigated. Diabetes was produced by the administration of streptozotocin, 60 mg/kg iv, and BP metabolism was studied 7 or 10 days later. BP metabolism was increased in hepatic microsomes by 75% in diabetic animals. Cytochrome P-450 levels were similarly increased. BP mono-oxygenase activity was tripled in intestinal microsomes of diabetic rats, and returned to control values on insulin treatment. The BP mono-oxygenase activity in lung microsomes from diabetic rats decreased by 40%, and was increased to control levels after insulin treatment. No significant changes in BP metabolism were observed in the kidney and adrenal tissues of diabetic animals.

Effect of cyanoketone on digitoxin-induced mortality and hepatic mixed function oxidases in rats.

Cyanoketone pretreatment protected female rats against digitoxin-induced mortality. Cyanoketone also acts as an inducer of hepatic mixed function oxidases, increasing cytochrome P-450 content and enhancing aniline hydroxylase and aminopyrine demethylase activities. The protective effect of cyanoketone against digitoxin toxicity may be due to the enhanced conversion of the glycoside to more polar metabolites which are more readily excretable.