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Concentrations of human growth hormone (HGH) in pituitary tumours measured by the tibial line assay (Young, Bahn & Randall, 1965) or assessed visually from electrophoretic patterns of pituitary extracts (Lloyd & Meares, 1965) have been shown to vary from quite low levels to concentrations greater than those present in the normal anterior pituitary gland. In the present study, HGH concentration in four normal anterior pituitary glands, obtained at autopsy, and in six pituitary tumours, resected surgically, has been estimated by radioimmunoassay and by inspection of starch-gel electrophoresis patterns. Histological examination of the tumour tissue was performed on paraffin sections of formalin-fixed specimens after staining with haematoxylin and eosin, eosin and methyl blue, and PAS-orange G. Extracts of pituitary tissue were prepared by homogenization in tris-EDTA-boric acid buffer at pH 8·9. The clear supernatant obtained after centrifugation was subjected to radioimmunoassay and to starch-gel electrophoresis. Radioimmunoassay was performed by the solid-phase
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ABSTRACT
The multifactorial control of ACTH is well established. We wished to establish and characterize an in-vitro perifusion system, using equine anterior pituitary cells and physiological concentrations of secretagogues, to investigate factors which affect the dynamics of ACTH secretion. Anterior pituitary tissue was divided for dispersion into cells with collagenase, trypsin or dispase, or by mechanical dispersion. After dispersal followed by 18-h incubation, cells were perifused and the ACTH response to 10-min pulses of arginine vasopressin (AVP; 100 nmol/l), corticotrophin-releasing hormone (CRH; 0·01 nmol/l), and AVP (100 nmol/l) plus CRH (0·01 nmol/l) determined. ACTH responses to these secretagogues were lower (P <0·05) in cells prepared using the enzymes dispase and trypsin than with the enzyme collagenase. Cells prepared by mechanical methods were not responsive. Collagenase-prepared cells were used in subsequent experiments.
In dose-response studies (10-min pulse length), a steep CRH–ACTH dose-response curve was obtained with the minimum effective concentration of CRH between 0·001 and 0·01 nmol/l, and a maximum effective concentration of 1·0 nmol/l. A less steep AVP–ACTH dose-response curve was obtained with a minimum effective concentration of AVP between 0·5 and 5 nmol/l, and no plateau in response up to 5000 nmol AVP/l. Increasing the incubation time between cell preparation and stimulation with AVP from 18 h to 90 h significantly (P <0·01) increased the ACTH response. Repeated stimulation by AVP (100 nmol/l) or CRH (0·01 nmol/l) (5-min pulses every 30 min for 23 pulses) produced ACTH responses which decreased in an approximately exponential curve with time.
When AVP and CRH were given at physiological concentrations, pulse lengths and pulse frequency, the ACTH response to repeated 1-min pulses of AVP, measured as height above basal secretion, was potentiated by the addition of CRH (1, 2·5, 5, 10 and 20 pmol/l) as a constant perifusion at all AVP concentrations tested (1 nmol AVP/l, P < 0·02; 10 nmol AVP/l, P <0·0005; 25 nmol AVP/l, P <0·0005). During the 1-min AVP pulse, the AVP concentration at the level of the cells was 30% of the expected concentration. Potentiation was increased both by increasing AVP concentration (P <0·00005) and by increasing CRH concentration (P <0·00005) up to 5 pmol CRH/l. The ACTH height response to repeated AVP stimulation significantly (P = 0·0034) decreased with time, independent of CRH and AVP concentration. There was a significant (P = 0·014) decrease in ACTH response to CRH infusion with time, independent of CRH concentration.
We conclude that the responsiveness of pituitary cells is markedly influenced by the preparative techniques. The collagenase-dispersed cells, in the in-vitro perifusion system developed, responded to secretagogues which were given at physiological concentrations, pulse lengths and periods. The system thus fulfills our requirements of in-vitro responses reflecting those observed in vivo, and can therefore be used to investigate the multifactorial control of ACTH secretion further.
Journal of Endocrinology (1993) 137, 391–401
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First morning urine samples were collected from both menstruant and post-menopausal women and stored at −25 °C. Immunoreactive FSH disappeared from these samples (t½ = 30 days), ultimately stabilizing at about 20% of the initial value. The loss was more rapid at −20 °C and less rapid at −55 °C and +4°C. Immunoreactive LH was also lost from frozen urine, but more slowly than FSH. The addition of glycerol to urine (0·52 mol/l) stored at −25 °C prevented loss of immunoreactive FSH and LH for at least 105 days.