(294) 6-NITRODOPAMINE IS AN ENDOGENOUS MEDIATOR OF RAT SEMINAL VESICLES CONTRACTILITY

Abstract Introduction 6-Nitrodopamine (6-ND) is released from rat and human isolated vas deferens and acts as an endogenous modulator of vas deferens contractility. Objective To investigate whether rat isolated seminal vesicles (RISV) present basal release of 6-ND, the mechanisms involved in the release, and the role of 6-ND on tissue contractility. Methods The local ethics committee approved the experimental protocols. Adult male Wistar rats (280 to 320 g) were used to perform the experiments. After isoflurane (5% for 2 min) induced euthanasia, the tissues were removed and one pair placed in 3 mL of Krebs–Henseleit’s solution at 37°C for 30 min, then an aliquot was used to analyze the concentrations of 6-ND, dopamine, noradrenaline, and adrenaline by Liquid Chromatography with tandem mass spectrometry (LC–MS/MS). The effects of the mechanical removal of the epithelium, the chronic treatment with L-NAME (20 mg/rat/day) in the drinking water and the pre-incubation of the RISV with L-NAME (100 μM), tetrodotoxin (1 μM), GKT137831 (1 μM) and H2O2 (100 μM) were also evaluated by LC–MS/MS. For functional studies, a RISV strip was mounted vertically in a glass chamber tissue bath containing 10-mL KHS with a fine sewing thread, one end attached through a loop to a metal hook and the other end tied to an isometric force transducer under a resting tension of 10mN and the isometric tension was registered using a PowerLab system (ADInstruments, Sdney, Australia). After tissue viability verification with noradrenaline (10 μM), concentration-response curves with 6-ND and classical catecholamines were performed, in addition interaction protocols among those drugs, including pre-incubation with tetrodotoxin, were assessed. Lastly, the expression of tyrosine hydroxylase was investigated by both immunohistochemistry and fluorescence in-situ hybridization. Results 6-ND was the major released catecholamine compared with noradrenaline, adrenaline, and dopamine (3.2 ± 0.7, 0.6 ± 0.4, 0.2 ± 0.1 and 0.3 ± 0.1 ng/mL, respectively). Mechanical removal of the epithelium caused significant reduction in 6-ND basal release, this depletion was also observed with the chronic treatment and the pre-incubation with L-NAME. In contrast, pre-incubation with either GKT137831 or H2O2 caused selective increases in the 6-ND release. Pre-incubation with tetrodotoxin had no effect on the release of catecholamines. All catecholamines produced concentration-dependent RISV contractions, but 6-ND was 30x less potent than the others. However, at low concentration (0.1 nM), 6-ND significantly potentiated noradrenaline-, adrenaline- and dopamine-induced contractions, which were not observed in presence of tetrodotoxin. Immunohistochemistry and fluorescence in-situ hybridization revealed the presence and expression of tyrosine hydroxylase in RISV epithelial cells. Conclusions This is the first demonstration of 6-ND release from epithelium and the results indicate that epithelial-derived 6-ND modulates rat seminal vesicle contractility. Disclosure No.