Regulation of corticotropin-releasing factor secretion from Leydig cells by serotonin

CRF is produced in the Leydig cells and acts as a negative autocrine regulator of Leydig cell function. To clarify the hormonal control of CRF secretion by Leydig cells, we evaluated the participation of serotonin (5HT) and serotonin agonists in the release of CRF from Leydig cells and their effects on hCG-induced cAMP generation and steroidogenesis. Serotonin stimulated CRF secretion up to 4-fold above basal levels and inhibited basal and hCG-stimulated cAMP generation and testosterone production (ID50, 1 nM). The inhibitory action of 5HT was prevented by a CRF antibody and the alpha-helical CRF-(9-41) antagonist. The selective 5HT2 receptor agonist (+-)1-[2,5-dimethoxy-4-iodophyryl]2-amino propane hydrochloride (DOI) also stimulated CRF secretion and inhibited hCG-stimulated cAMP generation and testosterone production to control levels (ID50, 7 microM). Serotonergic 5HT1A, 5HT1B/1C, 5HT1D, and 5HT3/5HT2 agonists were less effective inhibitors of hCG-stimulated cAMP and testosterone production, while agonists for the 5HT3 receptor had no effect. [125I]DOI binding studies in Leydig cells demonstrated two sets of receptors with Kd values in the nanomolar and micromolar range, with low and high capacities, respectively. The low affinity site differed from that of brain receptors (Kd, 4.2 nM) and displayed higher binding capacity (50-fold). The selective 5HT2 receptor antagonist ketanserin prevented CRF stimulation and blocked the inhibitory actions of 5HT and DOI, while the alpha 1-adrenergic antagonist prazosin had no effect. Also, treatment of cells with ketanserin increased sensitivity to hCG and raised maximal cAMP and testosterone production. 5HT was a more effective stimulus than hCG in stimulating CRF secretion, and gonadotropin-induced CRF release was inhibited by ketanserin. Inhibitory effects of exogenous CRF were demonstrable after blockade of 5HT action by ketanserin. The inhibitory actions of 5HT were unaffected by pertussis and cholera toxins and were reversed by the addition of 8-bromo-cAMP. These results demonstrate that 5HT acts on 5HT2 receptors in Leydig cells that are distinct from those in the brain to stimulate CRF secretion through a pertussis toxin-insensitive G-protein. This action of 5HT is predominantly mediated by the low affinity 5HT2-binding site and requires full occupancy for maximal CRF stimulation, indicating the absence of spare receptors. 5HT-stimulated CRF inhibits basal and hCG-induced cAMP generation and steroidogenesis. Furthermore, 5HT mediates the stimulatory action of LH/hCG on CRF secretion from Leydig cells and, thus, participates in a negative autoregulatory loop to limit the testosterone response to the gonadotropic stimulus.