Increasing saturation of the binding proteins following rises in testosterone production, and the small but significant changes in protein concentration, probably related to postural changes, were implicated as the major factors in the rhythm amplitude. However, the early morning decline in the non-SHBG-bound fraction was not explained by these factors. The rise in cortisol concentration at this time is a probable cause. Alternatively, simulation suggests that a substance appearing in the early morning and competing with testosterone for albumin binding sites may be responsible.

The effects were studied of varying the frequency, width and amplitude of pulses of gonadotrophin releasing hormone (GnRH) on the release of LH from anterior pituitary cells. Dispersed sheep cells supported in Sephadex were perifused with medium for 10 h and stimulated with different constant pulse patterns of GnRH. The timing of release of LH was measured by radioimmunoassay of the effluent fractions. Pulses of GnRH ranging in duration from 2 min every 8 min to 16 min every 128 min, and in concentration from 1.7 pmol/l to 250 nmol/l were applied to the cells, as well as continuous stimulation. Comparisons of differences between LH release patterns among samples of the same preparation of cells were used to demonstrate the effects of different GnRH stimulatory regimes. It was concluded that (1) the frequency of GnRH stimulation was important to the nature of LH release (periods shorter than about 16 min between pulses reduced LH output and caused faster desensitization of response), (2) the pulse width of GnRH input was important (the rising edge of the pulse produced greater LH output per unit of GnRH input than did continued application of GnRH within a pulse and wider pulses combined with shorter periods reduced LH output) and (3) over a threshold value of 5-10 nmol GnRH/l pulse amplitude had little further influence on LH output or rate of desensitization in dispersed cells. These findings reinforce the hypothesis that the rising edge of the GnRH pulse is the major stimulant to LH release.

No abstract

The dynamics of the release of proopiomelanocortin-derived hormones from ovine anterior pituitary cells in response to varying pulse characteristics of ovine corticotropin-releasing factor (CRF) were investigated with an in vitro automated 15-column simultaneous perifusion system. Columns of cells were stimulated continuously or with trains of CRF pulses of varying pulse length (2-16 min), pulse period (20-160 min), and concentration, for 500 min. Ovine ACTH, beta-lipotropin, and beta-endorphin immunoreactivity were measured by unextracted RIA. Each pulse of CRF stimulated clearly defined and highly correlated (r greater than 0.9) pulses of the three pituitary hormones, suggesting similar mechanisms controlling release. In dose-response experiments, the minimum concentration of CRF in a 10-min pulse required to significantly raise the output of ACTH was 200 pM, and initial responses had not attained maximal levels with concentrations of CRF increased to 2 microM. Responses to pulsed CRF stimulation decreased with time with all stimulation patterns selected, although previously unstimulated control columns retained the initial capacity to respond. Multiple linear regression analysis showed that hormone output per pulse of CRF (43 nM) increased with increasing pulse period and pulse length. Output of pituitary hormones per unit of CRF applied decreased with pulse length but increased with pulse period. In summary, the responses of proopiomelanocortin derivatives were shown to be sensitive to abrupt increases in CRF, to reduce output under continued stimulation, and to have an inherent time lag before responding maximally to subsequent pulsed stimulation.