The stimulation of somatostatin release by hpGRF44 from rat hypothalamic cells and fragments in vitro

Growth hormone releasing factor (GHRF) was examined to determine whether it affects somatostatin (SRIF) release from cultured rat hypothalamic cells and fragments in vitro. The hypothalami of rat fetuses were collected on the 17th day of pregnancy under a dissection microscope. Thirty hypothalami were placed in phosphate buffered saline, and the cells were dispersed with 0.1% collagenase. The dispersed cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% horse serum and 2.5% fetal calf serum at 37 degrees C under 5% CO2 in air. On the 12th day of culture, the cells were washed with Krebs Ringer bicarbonate buffer containing glucose (KRBG), and then incubated with KRBG for 1 hour. The medium was replaced with KRBG alone (control) or KRBG containing test substances, and incubated for another hour. SRIF released into the medium was quantitated by RIA. The mean basal release of SRIF was 14.7 +/- 0.9 pg/dish/hour. One-tenth, 1, and 10nM hpGRF44 stimulated SRIF release by 1.4, 1.5, and 1.8 fold respectively in a dose-related manner. Ten nM ovine corticotropin releasing factor (o-CRF) also stimulated SRIF release by 2.3 fold. One, 10, and 100 nM hpGRF44, 10nM o-CRF, 10nM thyrotropin releasing hormone (TRH) and 60 mM K+ also stimulated SRIF release from rat hypothalamic fragments. Removal of Ca++ from the medium resulted in a decrease of basal release of SRIF. In Ca++ free medium, 10nM hpGRF44 failed to release SRIF. One-tenth nM hpGRF44, 10nM GnRH, and 10nM VIP have no effect on SRIF release statistically. The results of this study demonstrate that a high concentration of GHRF stimulates SRIF release from the hypothalamus in vitro, suggesting a possibility that GHRF may increase the release of SRIF from the median eminence and the hypothalamus in vivo under certain conditions.