Long term superfusion of rat anterior pituitary cells: effects of repeated pulses of gonadotropin-releasing hormone at different doses, durations, and frequencies.

We investigated the temporal release of LH by rat pituitary cells superfused with repeated pulses of GnRH at different doses (amplitudes), frequencies, and pulse durations. Anterior pituitary (AP) cells prepared from ovariectomized rats were cultured on bio-beads and then placed in columns and superfused. The cells were stimulated intermittently for 24 h with synthetic GnRH given as pulses of 6-, 10-, or 15-min duration at frequencies of one pulse per 30, 60, or 90 min. In the first experiment we tested the temporal response of cells exposed to different doses of GnRH. At all doses, the first pulse of GnRH stimulated greater LH release than subsequent pulses. The response to subsequent pulses varied with dose. At low doses (1 and 5 X 10(-10)M) of GnRH, the AP cells stabilized and released similar amounts of LH in response to each sequential identical GnRH pulse for up to 24 h. In contrast, at high doses (10(-8) and 10(-6)M) of GnRH, the AP cells gradually released less LH with each pulse. During the initial pulses, the dose-response curves of LH release were linear between doses of 5 X 10(-10) to 1 X 10(-6) M GnRH. Thereafter, the maximum response was diminished, and the slope of the dose-response curve was reduced. In subsequent experiments we found that the decline in responsiveness with time was not due to either degradation of GnRH in the medium or loss of cell viability. The low responsiveness to high GnRH pulses at the end of the superfusion period could be overcome temporarily by further increasing the dose of GnRH. In the presence of high doses, changing GnRH pulse frequency from one 6-min pulse per h to one 6-min pulse per 1.5 h or changing duration from one repeated 10-min pulse per h to one repeated 6-min pulse per h had no detectable effect on the temporal pattern of LH release. These results suggest that, similar to constant infusion, pulsatile delivery of GnRH at high doses to AP cells can induce cell refractoriness to GnRH. This loss of responsiveness probably involves factors in addition to depletion of GnRH receptors and the releasable LH pool. In contrast, pulsatile delivery of low doses of GnRH appears to maintain LH release at a relatively constant level for up to 24 h.