Abstract
Abstract
Continuous perifusion of dispersed anterior pituitary cells recently has been applied to the study of LH secretion. We undertook a series of experiments to determine whether or not fetal calf serum(FCS) is a necessary or beneficial addition to medium perifusing cells in culture up to 43.5 hr. Male Sprague-Dawley rats provided pituitaries for two perifusion systems (columns) set up in parallel. The cells in one column were perifused on both Day 1 (first 19.5 hr) and Day 2 (subsequent 24 hr) with Medium 199, while the cells in the other column were perifused with Medium 199 on Day 1 and with Medium 199 with 2% FCS for the initial 18 hr on Day 2. On Days 1 and 2 cells in both columns were challenged with five doses of GnRH (1-100 nM) given as 1.5-min pulses in a random order. After 4-hr of perifusion, basal release of LH decreased gradually with time in a manner that fit a least-squares derived second-order polynomial function. Mean basal LH release (ng/ml/107 cells ± SEM) on Days 1 (9822 ± 2450) and 2 (7710 ± 2100) was no different (P = 0.1). Mean GnRH-stimulated LH release on Day 1 (1063 ± 144.3) was higher than on Day 2 (746 ± 143.7) (P = 0.036). GnRH-stimulated LH release on Day 2 was the same for the columns with (805.9 ± 41.9) and without (653.2 ± 190.8) FCS (P = 0.9); furthermore, the slopes of the dose-response curves were identical (P = 0.9). Thus, our results showed that, using continuously perifused dispersed rat anterior pituitary cells for up to 43.5 hr: (1) basal LH release does not change significantly; (2) GnRH-stimulated LH release becomes less with time but dose-response characteristics remain constant; and (3) preincubation with FCS appears unnecessary to maintain responsivity of cells. Furthermore, we propose that the quantitative methods used to analyze our data may prove useful to other investigators employing similar perifusion techniques, thus allowing comparison of results from various laboratories.
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