The role of the cAMP/PKA signalling pathway in the inhibitory influence of melatonin on oxytocin and vasopressin secretion from the rat hypothalamo-neurohypophysial system.

INTRODUCTION: Melatonin was found to inhibit forskolin-stimulated oxytocin (OT) and vasopressin (VP) release in vitro. The purpose of the present investigation was to evaluate the contribution of the cyclic 3',5'-adenosine monophosphate/protein kinase A (cAMP/PKA) signalling pathway in melatonin-dependent inhibition of OT and VP secretion from the rat hypothalamo-neurohypophysial (H-NH) system in vitro. MATERIAL AND METHODS: The H-NH explants were placed in 1 ml of normal Krebs-Ringer (nK-R) buffer and first preincubated for 30 min in control buffer or in the presence of PKA inhibitor, i.e. cAMPS-Rp or H-89. Next, they were incubated in nK-R buffer {fluid F1} and then in buffer as F1 enriched with melatonin (10-9 M or 10-7 M) and/or PKA activator, i.e. cAMP analogue (8-Br-cAMP), or their vehicles {fluid F2}. After 20 min of incubation in fluid F1 and then F2, the media were collected and frozen, to be assayed for OT and VP by the RIA. RESULTS: 8-Br-cAMP increased OT and VP secretion when the H-NH explants were preincubated in control medium, while PKA inhibitors eliminated its stimulatory effect on OT and VP release. Melatonin (10-7 M) diminished basal OT and VP output from the H-NH system, and inhibited (at both concentrations studied) the cAMP analogue-stimulated release of both neurohormones under control conditions. The effect of melatonin on OT and VP release was completely blocked when cAMPS-Rp, but not H-89, was used to disrupt the cAMP/ /PKA pathway. CONCLUSIONS: Melatonin employs the cAMP/PKA signalling pathway to inhibit OT and VP secretion from the rat H-NH system; nonethe-less, other cAMP-mediated mechanisms are not excluded.

Large deletion in the KAL1 gene in two related patients with hypogonadotropic hypogonadism: diagnostic usefulness of cytogenetic and molecular methods.

BACKGROUND: Kallmann syndrome type 1 (KS1) is a heterogeneous disorder where hypogonadotropic hypogonadism (HH) associated with an impaired sense of smell is observed. The aim of this study was to investigate the usefulness of the multiplex ligation-dependent probe amplification (MLPA) technique for differential diagnosis in comparison with molecular cytogenetics - fluorescence in situ hybridisation (FISH) or traditional PCR analysis and propose a diagnostic approach for patients with KS. MATERIAL AND METHODS: Karyotype and PCR analysis in two related patients and other family members were performed, followed by MLPA dosage sensitive analysis. RESULTS: In the proband and his maternal uncle, the PCR allowed the detection of a large deletion within the KAL1 gene, from exon 4 to 14 (c.469-?_6314+?del). The deletion was also diagnosed in three female carriers in the presented family. These results were proved by the MLPA technique. Moreover, we traced the presence of the region located downstream and upstream to the KAL1 gene on Xq22.32. However, FISH analysis failed to reveal any deletion in the critical region for KS. Simultaneously, we report difficulties connected with the PCR technique based on the primers for KAL1 amplification presented in the literature. We designed primers that are specific to the X chromosome and bypass pseudogene KALY amplification. CONCLUSIONS: FISH analysis is a convenient screening technique, but in the presented family it failed to detect the deletion. Therefore, in the face of a distinctive manifestation of KS, a subsequent molecular assay should be introduced. The MLPA is a useful technique for differential diagnosis in patients with HH combined with smell impairment.