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1 The endothelium-dependent relaxation of blood vessels induced by P2y-purinoceptor activation has often been shown to involve prostacyclin and/or nitric oxide (NO) release. In this work, we have investigated the mechanisms involved in the relaxant effect of the P2y agonist, adenosine -5'-O-(2-thiodiphosphate) (ADPflS) using two complementary preparations: rat pancreatic vascular bed and aortic ring.2 On the pancreatic vascular bed, ADPPS (1.5 and 15 gM) infused for 30 min induced a concentrationdependent vasodilatation; it was progressive during the first 10 min (first period) and sustained from 10 to 30 min (second period). Indomethacin (10 gM) delayed ADPBS-induced vasodilatation (1.5 and 15 guM) by about 6 min. N()-nitro-L-arginine methyl ester (L-NAME) (200 gM) suppressed the relaxation for about 5 min but thereafter ADPBS at the two concentrations progressively induced an increase in the flow rate. Even the co-administration of L-NAME and indomethacin did not abolish the ADPPSinduced vasorelaxation. 3 On 5-hydroxy tryptamine (5-HT) precontracted rings mounted in isometric conditions in organ baths, we observed that ADPPS induced a concentration-dependent relaxation of rings with a functional endothelium; this effect was stable for 25 min. The ADPPS relaxant effect was strongly inhibited by Reactive Blue 2 (30 gM) and was suppressed by pretreatment of rings with saponin (0.05 mg ml-' for 30 min), which also abolished the acetylcholine-induced relaxation. 4 ADPBS-induced relaxation of 5-HT precontracted rings is largely inhibited by indomethacin (100 or 10 pM) or L-NAME (100 gM). 5 We conclude that: the ADPBS-induced relaxation is endothelium-dependent, mediated by P2Y-purinoceptors, and at least in part linked to NO and prostacyclin release, depending on the preparation used. Furthermore, on the pancreatic vascular bed, (an)other mechanism(s) than prostacyclin and NO releases may be involved in ADPPS-induced vasodilatation.

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The effects of melatonin on LH and PRL releases induced by treatment with naloxone, naloxone methyliodide and nalmefene were studied in adult male rats. Subcutaneous melatonin injection (1.4 mg/Kg) had no effect on LH secretion, but caused an inhibition effect (84%) on LH release induced by naloxone (2.4 mg/Kg). Melatonin too totally inhibited LH secretion induced by naloxone methyliodide (2.8 mg/Kg) and nalmefene (2 mg/Kg) when it was simultaneously administered with each opioid receptor antagonist. Melatonin alone had no significant effect on serum PRL levels, but decreased by 25.5% the inhibitory effect potency of nalmefene on PRL secretion after simultaneous injections. The inhibitory effect potency of naloxone on PRL release increased (16%) when it was administered with melatonin. Simultaneous injection of melatonin with naloxone methyliodide inhibited PRL release (78%) while naloxone methyliodide alone did not modify this secretion. The results obtained with a quaternary opioid antagonist indicate that the opioid receptor type which mediates LH and PRL responses is located respectively outside and inside the blood-brain barrier. Our findings show that opiate antagonists and their quaternary ammonium salts affect secretion of LH and PRL through different mechanisms susceptible to the influence of melatonin.

Three dimensional (3-D) cultures of pineal cell aggregates were obtained by constant gyratory shaking the heterogenous cell populations, obtained from the rat pineals, in the DMEM (Dulbecco's modified Eagle's medium). Within 4 days, the pineal cells became organized into a tissue like configuration appearing as a compact ball, evidenced by the scanning electron microscopy. The 3-D aggregates seemed to be mainly composed of pinealocytes (round-oval cells), glial (elongated cells) and other unknown cells. The heterogenous cells were separated by intercellular spaces. The ultrastructural characteristics revealed by transmission electron microscopy exhibited the presence of granular lysosomes, typical of pinealocytes actively involved in the secretion. These pineal cell aggregates secreted melatonin and other indole amines i.e. 5-methoxytryptamine (5-MT), indole acetic acid (IAA), 5-methoxy-3-indole acetic acid (5-MIAA), tryptophol (TOL) and 5-methoxytryptophol (5-MTL) in the culture medium, indicating the functional aspect of pinealocytes. The 3-D aggregates cultures had advantages over the pineal monolayer cultures as, after 4 days of culture, the amounts of indole amines secreted by 3-D aggregates were higher than those secreted by monolayer cultures. Besides, the 3-D aggregates remained functional till 24 days in the gyratory culture conditions. In the continuous perifusion system, the 3-D aggregates secreted melatonin while challanged with isoproterenol. This 3-D model of pineal cell aggregates might be useful, in future, to perform other kinetic studies of the release of indole amines in perifusion experiments as this system allows the maintenance of pineal cells for a long period of time.