TYPE-1 ASTROCYTES INFLUENCE LUTEINIZING - HORMONE-RELEASING HORMONE-RELEASE FROM THE HYPOTHALAMIC CELL-LINE GT1-1 - IS TRANSFORMING GROWTH-FACTOR-BETA THE PRINCIPLE INVOLVED

The possible existence of a humoral communication between glial cells and LHRH-secreting neurons has been studied using the LHRH-secreting G T1-1 cell line and type 1 astrocytes. Two different designs have been adopted: 1) GT1-1 cells were coincubated with purified cultures of typ e 1 rat astrocytes, and 2) GT1-1 cells were exposed to the conditioned medium (CM) in which type 1 rat astrocytes had been grown for 24 h. L HRH was measured by RIA in the medium of the GT1-1 cell cultures at di fferent time intervals. The data show that short periods (1, 3, and 6 h) of either coculture or exposure to previously frozen CM significant ly increase the release of LHRH from the GT1-1 cells. However, more pr olonged times of coculture (e.g. 2 and 5 days) or exposure to CM (e.g. 48 h) induce a significant decrease in the amount of LHRH in the medi um. The stimulatory effect on LHRH release appears to be specific for type 1 astrocytes (either cortical or hypothalamic), because neither t he CM, of oligodendrocytes nor the CM of LNCaP cells (a cell line deri ved from a human prostatic cancer) possess stimulating activities. Hea ting the type 1 astrocyte-CM to 100 C for 10 min does not eliminate th e ability of the CM to significantly increase the release of LHRH from GT1-1 cells at 1, 3, and 6 h. Because of the opposite effects encount ered in the short and long term experiments, it was hypothesized that the CM might contain, in addition to LHRH-releasing principle(s), LHRH -degrading properties. Known amounts of standard LHRH were then added to type 1 astrocyte-CM, either untreated or submitted to heating at 10 0 C for 10 min. The amount of LHRH added to untreated CM decreases pro gressively; on the contrary, the amount of LHRH added to heated CM rem ains unchanged. These results confirm that one or more heat-sensitive enzymes able to degrade LHRH may be present in the type 1 astrocyte-CM . As previously mentioned, the experiments reported so far were perfor med using type 1 astrocyte-CM that had been kept frozen for various pe riods of time, before being tested for its LHRH-releasing activity. Su rprisingly, fresh CM proves to be inactive, whereas heated CM is effec tive; this suggests that the factor involved might be activated by the two opposite experimental procedures. As it is known that transformin g growth factor-beta (TGF beta) may be liberated from its supporting p roteins by freezing or heating, the effects of TGF beta and a TGF beta -neutralizing antibody were analyzed. The stimulatory effect exerted b y the frozen CM is completely abolished by 5 mu g of the antibody, whe reas 5 ng TGF beta 1 proves able to significantly increase LHRH releas e from GT1-1 cells. In conclusion, the present experiments demonstrate that type 1 astrocytes of either cortical or hypothalamic origin may secrete in the medium some factor(s) (probably TGF beta) able to stimu late the release of LHRH from the hypothalamic LHRH-producing GT1-1 ce lls; moreover, the data show that one or more enzymes degrading LHRH m ay be present in type 1 astrocyte-CM. The data provide the first clear -cut demonstration that glial cells may directly intervene in the cont rol of LHRH release, integrating the old concept that only neuronal in fluences may participate in such a phenomenon.