The Adenylyl Cyclase‐Cyclic AMP System Modulates Morphological and Functional Development of Hypothalamic β‐Endorphin Neurons in Culture

In rats, opioidergic β‐endorphin (βEP_1–31) is produced and released from neurons of arcuate nuclei in the hypothalamus. Although the neuropeptide has been implicated in sexual maturation and stress‐induced reproductive dysfunction, the intra‐hypothalamic regulation of βEP neurons remains unclear. Employing long‐term monolayer cultures of neonatal rat hypothalamic cells, we report here that 4 days of treatment with 10 μM forskolin increased approximately 3‐fold (P<0.01) the proportion of immunoreactive (ir)‐ βEP positive neurons bearing neurites. In addition, treatment of forskolin also enhanced ir‐βEP release (634 ± 59 pg/well; mean β SE, n = 4, P<0.01) by 14‐fold and ir‐βEP content (119±13 pg/well; P<0.01) by 2‐fold above that of vehicle‐treated cultures; in both instances, the EC₅₀ and the E_max, of forskolin were approximately 10μM and 100 μM, respectively. The forskolin‐stimulated release of ir‐βEP was mimicked by cholera toxin and (Bu)₂cAMP treatment in a dose‐related manner, but not by pertussis toxin. Although by itself 3‐isobutyl‐1‐methyl‐xanthine (100μM) only doubled ir‐βEP secretion, it markedly potentiated the stimulatory effect of forskolin. This forskolin‐induced stimulation was reversible and in cultures re‐exposed to the same drug within the first 24 h period, there was a marked increase in the stimulated release of ir‐βEP (P < 0.05); re‐challenge of forskolin at later stages, however, induced a smaller but significant secretion of ir‐βEP (P<0.01) compared to that of vehicle‐treated control cultures. Sephadex G‐50 gel Chromatographie profile of the media prepared from forskolin‐treated cultures revealed a major ir‐βEP peak of 3 K M. Highperformance liquid chromatography analysis showed that ir‐βEP of the 3 K M, species was eluted with a retention time similar to that of synthetic rat βEP_1–31. We thus conclude that the adenylyl cyclase‐cAMP system plays an important role in the modulation of βEP_1–31 production and release from hypothalamic βEP neurons in culture. Furthermore, the functional responsiveness and the orphological development of these neurons are affected, at least in part, by the intrinsic activity of the adenylyi cyclase‐cAMP system.