GROWTH HORMONE-RELEASING FACTOR REGULATION BY SOMATOSTATIN, GROWTH-HORMONE AND INSULIN-LIKE GROWTH-FACTOR-I IN FATAL RAT HYPOTHALAMIC-BRAINSTEM-CELL COCULTURES

Information about growth hormone-releasing factor (GRF) regulation by somatostatin, GH and IGF-I is scarce and controversial. This could be due to the in vivo interactions among these signals and the lack of mo dels for individualizing the action of one of them from the others upo n GRF regulation. The aim of the present work was to study GRF regulat ion by these signals, using primary fetal rat hypothalamic-brain stem cell cocultures. Coculturing of these two cytotypes increases hypothal amic immunoreactive rat GRF (IR-rGRF) content in cells by 45% and in m edia by 36%. The effect of SS on GRF in cocultures was examined by usi ng a multiple approach: (1) depleting endogenous SS by adding 1 mM cys teamine (CSH); (2) blocking endogenous SS by incubation with SS antise rum, and (3) incubating with synthetic SS14 at different concentration s and exposure periods. 1 mM CSH depleted IR-SS content (pg/plate, mea n +/- SE) in cells (CSH-treated: 68 +/- 8 vs. control: 322 +/- 10, p<0 .01) and media (CSH-treated: 211 +/- 15 vs. control: 880 +/- 70; p<O.O 1). In the CSH-induced SS-depleted cultures, a slight reduction in the IR-rGRF content in cells was observed (CSH treated: 93.5 +/- 4.5 vs. control: 111 +/- 6; p<0.05), with no effect on media content. When SS antiserum was added to plates, there was a slight reduction in the IR- rGRF content in cells and media, but it was not significantly differen t from the controls. However, SS14 (10(-10) -10(-8) M) could not modif y IR-rGRF content in media and cells. The GH effect on IR-rGRF was stu died in the absence of CSH and in CSH-induced SS-depleted cultures. GH (5 mu M, 24 h) decreased (52%) the IR-rGRF content in media (GH-treat ed: 28.7 +/- 4.6 vs. control: 60.2 +/- 7; p<0.01) without causing chan ges in cell content. In SS-depleted cultures, the inhibitory action of GH on media IR-rGRF was greater (62% decrease) (GH-treated: not detec ted, control: 56 +/- 10; p<0.01) and also affected IR-rGRF cell conten t (GH-treated: 64.3 +/- 7.3 vs. control: 160 +/- 9.6; p<0.01). In the same experiments, GH increased IR-SS content in cells (GH-treated: 31. 8 +/- 4.6 vs. control 20.9 +/- 0.5; p<0.01) and in media (GH-treated: 413 +/- 7 vs. control: 286 +/- 9; p<0.01). 1 mM CSH again depleted IR- SS content and abolished the GH stimulatory effect. IGF-I (5.6 nM, 24 h) did not affect IR-rGRF content in media or in cells under either co nditions (CSH and non-CSH-treated cultures). However, in these experim ents IGF-I increased IR-SS content in the media (GH-treated: 322 +/- 3 vs. control: 289 +/- 9; p<0.05). These results indicate that: (1) Coc ultures of brain stem and hypothalamic cells exert a positive action o n the hypothalamic GRF; (2) locally secreted SS does not have an impor tant role in the regulation of GRF in our experimental model; (3) GH h as a direct inhibitory effect on GRF secretion which is independent of the SS released by GH; (4) IGF-I does not affect GRF at the concentra tion used in the experiment, and (5) CSH-treated fetal rat hypothalami c-brain stem cocultures provide information potentially useful in unde rstanding GRF regulation independently from that of SS.