A superfusion system technique for the study of the sites of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. I. Pituitary cell superfusion.

Rat anterior pituitary cells were placed in a superfusion column and were stimulated by rat hypothalamic extract. The rate of ACTH secretion was monitored by collecting fractions eluting from the column every 2 min. Dispersed rat adrenocortical cells were used to determine the amounts of ACTH in the superfusate. ACTH appeared in the medium 6-12 sec after stimulation of the cells. A linear log dose-response relationship existed between 1/8 to 1 rat hypothalamus extract and the amounts of ACTH released. Repeated pulses of CRF stimulation of the same column of pituitary cells resulted in repeated, identical peaks of ACTH. Constant stimulation caused a plateau in hormone release that lasted as long as the stimulus was present. The stimulated, but not the basal ACTH release, could be inhibited by superfusion with corticosterone in vitro (0.2 mug/ml), or by treating the pituitary donor animal in vivo with corticosterone (5 mg/100g BW), cortisol or dexamethasone. Adrenalectomy of the donor animal caused increases in the basal and the stimulated ACTH release.

A superfusion system technique for the study of the sites of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. II. Hypothalamus-pituitary cell-adrenal cell superfusion.

Basal and stimulated CRF release by hypothalamic blocks was studied by coupling the effluent of superfused hypothalamus tissue to a joint pituitary cell-adrenal cell superfusion system and measuring corticosterone production. Log dose-response curves of the adrenal cells for ACTH and of the pituitary cell-adrenal cell system for CRF were linear over the ranges used. Ca++-independent basal CRF release by the hypothalamus could be blocked in vitro by 0.2 mug/ml dexamethasone in the medium, or in vivo by treating the hypothalamus donor rats with corticosterone, 1 mg/rat ip 30 min before decapitation. These treatments did not impair CRF release caused by Veratridine (5 x 10(-6)M or by electrical stimulation. Adrenalectomy increased only basal but not stimulated CRF release. These results indicate that glucocorticoids have a hypothalamic site of action.

Luteinizing hormone secretion patterns in boys at the onset of puberty measured using a highly sensitive immunoradiometric assay.

Pulsatile LH secretion was studied in 3 prepubertal and 11 early pubertal boys by measuring plasma LH concentrations at 10-min intervals from 1200-1800 h and from 2400-0600 h using an immunoradiometric assay with a lower limit of detection of 0.10 IU/L. Plasma testosterone (T) was measured hourly. In the prepubertal boys plasma LH was not detectable during the daytime but at night 20- to 300-min periods of detectable, but low (less than 0.5 IU/L) plasma LH values occurred. A discrete episodic LH pattern was discernible, and the median number of pulses was 2 during the 6-h nocturnal sampling periods. Plasma T was not detectable (less than 1.0 nmol/L). In the pubertal boys most daytime plasma LH values were greater than 0.3 IU/L, with periods of values of 0.1-0.3 IU/L and short periods of undetectable levels as well. At night definite pulses, up to 4.7 IU/L, were found in all boys. The median number of pulses was 4 during the 6-h nocturnal sampling period. Plasma T was detectable at night in 5 of these 11 boys. The results strongly suggest that at the onset of puberty prepubertal boys (G1) have no LH secretion during the day but intermittent gonadotrophic activity during the night. In early puberty LH secretion increases in amplitude as well as frequency to a clear pulsatile pattern during the night, sometimes with pulses during the day as well.

Hypothalamic corticotrophin releasing factor activity in dogs with pituitary-dependent hyperadrenocorticism.

Hypothalamic corticotrophin releasing factor (CRF) activity was determined in five dogs with spontaneous hyperadrenocorticism and in three control animals (one untreated, one treated with high doses of ACTH for 2 months and one treated with high doses of cortisone for 2 months). Hypothalamic CRF activity was low or undetectable in four dogs with Cushing's syndrome due to an adrenocortical tumour. The results are compatible with a pituitary origin for pituitary-dependent hyperadrenocorticism in the dog but are not conclusive; direct information about the rates of hypothalamic CRF secretion is required.

In vitro secretion of peptide hormones by the human placenta: I. ACTH.

In order to address the question of autonomy of placental hormone secretion, fresh human term placentae were utilized for the preparation of small tissue fragments. The fragment pool was divided over four parallel chambers in a superfusion apparatus and could thus serve as both control and experiment under identical in vitro conditions. Oxygen consumption was substantial and could be maintained for at least 5 h. Adrenocorticotrophic hormone (ACTH) concentrations in the effluent buffer were estimated by radioimmunoassay and bioassay. Both non-specific (membrane depolarization with 45 mM KCl) and specific (isoproterenol at 10(-7) M) stimulation increased the ACTH secretion from 10 to 20 pg/min/g to 60 to 80 pg/min/g. Propranolol blocked the adrenergic stimulation almost completely, indicating the specificity of the effect. Thus, in terms of in vitro ACTH secretion, the human placenta can be stimulated and therefore does not seem to behave in an autonomous manner.

Fluid transport across human fetal membranes affected by human amniotic fluid prolactin: an in vitro study.

The aim of the study was to investigate the effects of human amniotic fluid (AF) prolactin (PRL) on the transfer of fluids across human fetal membranes, detected in vitro by weight change. We have developed a system, consisting of an inner compartment (4 ml), suspended in an outer compartment (40 ml), and separated from it by a 2 cm2 circular piece of fresh human fetal membrane (amnion, chorion and adhering decidua). The weight of the inner compartment was continuously registered with an electronic precision balance. Osmolality changes or hydrostatic pressure differences did not affect the rate of mass transfer through the membranes. Potassium cyanide significantly influenced the mass transfer in favour of the applied hydrostatic pressure difference (+2 cm H2O to the amniotic side) or osmotic pressure difference (amniotic side 265 mosm/kg/decidual side 285 mosm/kg). The PRL preparations used were human and ovine pituitary PRL, as well as three fractions isolated from human AF by fractionated ammonium sulphate and ethanol precipitations, followed by Sephacryl chromatography. All PRL preparations were tested in physiologic concentrations (0.5 to 5 micrograms/ml). Only the two largest AF-PRL variants significantly disturbed the balance of fluid transfer across the membranes when added to the fetal (amniotic) side of the membrane. This resulted in a net increase in fetomaternal transfer of 120 to 180 microliters. This effect could be repeated and lasted for at least 25 minutes. Using an antibody against hPRL the effect was completely blocked. Neither AF-PRL added to the maternal (decidual) side of the membrane nor oPRL or human pituitary PRL added to the amniotic or decidual side changed the rate of mass transfer across the membranes.

Release of beta-lipotropin and beta-endorphin from rat hypothalami in vitro.

Hypothalamic tissue extracts of rats were chromatographed and beta-endorphin immunoreactivity (beta-Endi) was measured. The two major peaks of beta-Endi co-eluted with beta-lipotropin (beta-LPH) and beta-End respectively. Hypophysectomy caused a local decrease of beta-LPH and beta-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM k+ induced a Ca2+-dependent release of both beta-LPH and beta-End.

A combined cell column superfusion technique to study hypothalamus-pituitary-adrenal interactions in vitro.

Rat anterior pituitaries and rat adrenals were dispersed into cell suspensions. In a cell column superfusion system the pituitary cells were used to study ACTH release and the adrenal cells served as a means to detect that hormone. Linear log-dose-response relationships were exhibited by the adrenal cells for ACTH and by the pituitary cell-adrenal cell system for corticotropin-releasing factor (CRF). A combined system, constructed of a superfused hypothalamus, leading into a column of pituitary cells which was connected to a column of adrenal cells, was used to study the characteristics of CRF release from the hypothalamic block. Basal and stimulated CRF release were both Ca(2+)-dependent.

Effects of naloxone on hemodynamics, oxygen transport, and metabolic variables in canine endotoxin shock.

The effects of naloxone (2 mg/kg body weight) on hemodynamics, oxygen transport, and some metabolic variables were studied in mongrel dogs under general anesthesia and controlled ventilation. All 19 dogs received Escherichia coli endotoxin (1.5 mg/kg) and subsequently were randomized into three groups. The first group (N = 7) served as a control group in which at 90 min after endotoxin (t 90), NaCl (0.65%, 4 ml/kg) was infused during 30 min. In the second group (N = 7) naloxone (2 mg/kg) dissolved in the same amount of fluid was administered at t 90 in 30 min. In the third group (N = 5) naloxone (2 mg/kg) was injected as a bolus in 5 ml NaCl (0.65%) at t 90, which was followed by NaCl (0.65%, 4 ml/kg) infused in 30 min. Differences in the three groups after intervention were tested statistically. After naloxone, blood pressure, cardiac output, and left ventricular stroke work increased significantly. Although oxygen availability increased, oxygen consumption and serum lactate did not change when compared with the control group. As to all other measured and calculated variables, no systematic differences were found in the three groups. In six dogs, plasma beta-endorphins were measured and were shown to rise substantially after induction of endotoxin shock. As to the hemodynamic changes, our observations confirm data from the literature. Naloxone apparently improves hemodynamics in endotoxin shock, but at least in this study fails to influence oxygen consumption and serum lactate levels.

Sequence analysis of a split gene involved in fruiting from the fungus Schizophyllum commune.

The sequence of a gene and its mRNA, which is abundantly expressed during fruiting body initiation in the Basidiomycete Schizophyllum commune, is described. This gene (1G2), the first to be analyzed in this group of fungi, contains an open reading frame coding for a polypeptide of 94 amino acids and a mol. wt. of 9842. A possible signal peptide of 20 residues and one glycosylation site were found. The sequence analysis was hampered by a sequence rearrangement in one of the cDNA clones, probably due to base pairing between short complementary sequences present at the 5' and 3' ends of the mRNA. The 5' untranslated leader sequence is 57 bp long and harbors a possible ribosome binding site close to the AUG start codon. A TATA box is found at position -31 upstream of transcription initiation. The 3' untranslated sequence is 200 bp long and contains the sequence -TATATAAT-, which most likely represents the polyadenylation signal. Some heterogeneity as to the site of addition of the poly(A) tail was observed. The coding region of the gene is interrupted by three very small introns of 53, 49 and 49 bp, respectively. The 5' and 3' splice junctions are conserved: GTGAGT- and -AG-, respectively. Each intron contains a sequence complementary to the 5' end of the intron. These sequences are compared with internal conserved sequences in yeast and filamentous fungi with regard to their possible role in splicing.