This study investigated the importance of reorganization of cell components by cytoskeletal structures to the short-term dynamic changes in LH release from dispersed sheep pituitary cells in perifusion, when stimulated with different dynamic patterns of gonadotrophin-releasing hormone (GnRH). The changes in rate of LH release investigated were the initial response to GnRH, desensitization, change of dose–response during desensitization, and recovery of sensitivity between pulses of stimulation. Cytochalasin D and colchicine were used to modify microfilament and microtubule action respectively. To determine whether receptor movement after binding of agonist was involved in the altered responses, K+ and phorbol 12-myristate 13-acetate (PMA) were used as stimulants because they cause LH release independently of agonist-receptor interaction.
After 3 and 48 h culture on dextran beads and 2–3 h incubation in the presence and absence of 2–48 μmol cytochalasin D/l, or 8 or 250 μmol colchicine/l, aliquots of collagenase-dispersed sheep pituitary cells were stimulated at 37 °C in tubes or in a multicolumn perifusion system with 850 pmol GnRH/l, 109 mmol K+/l or 10 nmol PMA/l. Fractions of supernatant or effluent were collected at intervals and LH concentrations measured by radioimmunoassay. Control samples were treated in the same way but without stimulation.
Maximal, reversible enhancement of LH release over the first 20 min following stimulation with all secretogogues was observed after incubation of cells in 6 μmol cytochalasin/l. Desensitization behaviour, the supramaximal response, and the ability of cells to recover sensitivity to repeated pulses of GnRH were not altered by this modifier of microfilament polymerization at 6 or 24 μmol/ml. Colchicine at 8 μmol/l caused no changes in LH release. At 250 μmol/l, colchicine reduced the initial response of cells to GnRH stimulation but its action at this relatively high level may not be specific; there was no other major change in desensitization patterns, nor recovery of sensitivity to pulsed GnRH stimulation. Each treatment affected cellular responses similarly before and after culture.
From studying the details of the dynamics of the short-term responses of gonadotrophs, we conclude that transport of cell components involving microfilaments and microtubules is unlikely to be a major limitation on the rate of LH release during desensitization, the supramaximal response, or the recovery of sensitivity between pulses of GnRH. This suggests that biochemical reactions rather than physical translocation may be rate-limiting in these processes. In addition, although inhibition of microfilament action does appear to enhance the earliest observed response to stimulation of the LH-release mechanism, this occurs after protein kinase C activation and is probably not related to impairment of processes such as polymerization and sequestration of agonist-bound GnRH receptors because the effects are also observed with K+ and PMA, stimulants acting independently of agonist-receptor interaction.