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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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ABSTRACT
Perifused equine anterior pituitary cells were used to investigate the effect of cortisol on the ACTH response to pulses of corticotrophin-releasing hormone (CRH; 0·01 nmol/l) and arginine vasopressin (AVP; 100 nmol/l), given for 5 min every 30 min for 690 min and ACTH measured in 5-min fractions. At the fourth pulse of secretagogue (0 min), a constant perifusion with cortisol began (0 nmol/l (control), 100, 200, 500, 5000 and 50 000 nmol/l) and continued until the ninth pulse (150 min). For each pulse of secretagogue, the amount of ACTH (pmol) secreted in response to each pulse (ACTH response area), the highest concentration of ACTH (μg/l) measured after each pulse (peak height) and the mean ACTH in the three prepulse fractions (ACTH baseline) were determined. Data from control columns in each experiment were fitted by least squares to an exponential function to produce a mean control value for each end-point; results in all columns were expressed as a percentage of the mean control values.
The addition of cortisol had a highly significant negative effect on ACTH response area, peak height and baseline at all times from + 30 to + 240 min (columns given cortisol compared with the mean of control column values by t-test). Analysis of variance of the data showed that the higher the cortisol concentration, the quicker the ACTH response area (P = 0·0072) and peak height (P = 0·002) decreased to < 50% of mean control, and the greater the maximum percentage change (suppression) in ACTH response area (P <0·0001) and peak height (P <0·0001). The maximum percentage change (suppression) in base-line was independent of cortisol concentration.
At + 30 min after the start of cortisol perifusion, the ACTH response area in CRH columns was significantly lower than in AVP columns (P = 0·0088), and remained lower 90 min after the end of perifusion (P = 0·0084) but the maximum percentage change (suppression) was not different between secretagogues. ACTH peak height was significantly (P < 0·0268) lower in CRH than in AVP columns (from + 30 min until 180 min after the end of cortisol perifusion) and the maximum percentage change (suppression) was also greater (P = 0·0011) in CRH columns.
This study shows the effect of different concentrations of cortisol on CRH- and AVP-induced ACTH secretion by equine anterior pituitary cells, and the time-course for ACTH responses to be inhibited by, and recover from, cortisol perifusion. The highly significant inhibitory effect of cortisol on stimulated ACTH secretion was more apparent when CRH was the secretagogue than when AVP was the secretagogue. The significant inhibitory effect of cortisol on unstimulated baseline secretion of ACTH has not been described previously. These effects occur at physiological concentrations of secretagogues and cortisol. This suggests that, in vivo, circulating cortisol may have an important role in the control of ACTH secretion at pituitary level.
Journal of Endocrinology (1993) 137, 403–412
