REGULATION OF CORTICOTROPIN-RELEASING FACTOR SECRETION FROM LEYDIG-CELLS BY SEROTONIN

CRF is produced in the Leydig cells and acts as a negative autocrine r egulator of Leydig cell function. To clarify the hormonal control of C RF secretion by Leydig cells, we evaluated the participation of seroto nin (5HT) and serotonin agonists in the release of CRF from Leydig cel ls and their effects on hCG-induced cAMP generation and steroidogenesi s. Serotonin stimulated CRF secretion up to 4-fold above basal levels and inhibited basal and hCG-stimulated cAMP generation and testosteron e production (ID50, 1 nM). The inhibitory action of 5HT was prevented by a CRF antibody and the alpha-helical CRF-(9-41) antagonist. The sel ective 5HT2 receptor agonist (+/-)1-[2,5-dimethoxy-4-iodophyryl]2-amin o propane hydrochloride (DOI) also stimulated CRF secretion and inhibi ted hCG-stimulated cAMP generation and testosterone production to cont rol levels (ID50, 7-mu-M). Serotonergic 5HT1A, 5HT1B/1C, 5HT1D, and 5H T3/5HT2 agonists were less effective inhibitors of hCG-stimulated cAMP and testosterone production, while agonists for the 5HT3 receptor had no effect. [I-125]DOI binding studies in Leydig cells demonstrated tw o sets of receptors with K(d) values in the nanomolar and micromolar r ange, with low and high capacities, respectively. The low affinity sit e differed from that of brain receptors (K(d), 4.2 nM) and displayed h igher binding capacity (50-fold). The selective 5HT2 receptor antagoni st ketanserin prevented CRF stimulation and blocked the inhibitory act ions of 5HT and DOI, while the alpha(1)-adrenergic antagonist prazosin had no effect. Also, treatment of cells with ketanserin increased sen sitivity to hCG and raised maximal cAMP and testosterone production. 5 HT was a more effective stimulus than hCG in stimulating CRF secretion , and gonadotropin-induced CRF release was inhibited by ketanserin. In hibitory effects of exogenous CRF were demonstrable after blockade of 5HT action by ketanserin. The inhibitory actions of 5HT were unaffecte d by pertussis and cholera toxins and were reversed by the addition of 8-bromo-cAMP. These results demonstrate that 5HT acts on 5HT2 recepto rs in Leydig cells that are distinct from those in the brain to stimul ate CRF secretion through a pertussis toxin-insensitive G-protein. Thi s action of 5HT is predominantly mediated by the low affinity 5HT2-bin ding site and requires full occupancy for maximal CRF stimulation, ind icating the absence of spare receptors. 5HT-stimulated CRF inhibits ba sal and hCG-induced cAMP generation and steroidogenesis. Furthermore, 5HT mediates the stimulatory action of LH/hCG on CRF secretion from Le ydig cells and, thus, participates in a negative autoregulatory loop t o limit the testosterone response to the gonadotropic stimulus.