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In a previous study we ascertained the presence of 1 and 2 recognition sites in the rabbit iris-ciliary body, an ocular structure involved in aqueous humor production and drainage. We characterized the 1 sites using the preferential ligand (ϩ)-pentazocine, which caused a significant reduction of intraocular pressure (IOP). In the present study, flunarizine, a calcium channel blocker with a complex pharmacological profile, bound to 1 sites expressed in the iris-ciliary body with moderate affinity (K i ϭ 68 nM). Unilateral topical flunarizine (0.01-0.1%) caused a dose-related reduction of IOP in ocular normotensive rabbits and in the ␣-chymotrypsin model of ocular hypertension, without altering the IOP of the contralateral eye. This activity was blocked by the 1 site antagonist NE-100 [N,Ndipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl] which, by itself, had no effect on IOP. Detection of flunarizine in rabbit iris-ciliary body homogenates, after topical instillation, showed that it adequately penetrates the rabbit eye. To investigate mechanisms that may contribute to ocular hypotension induced by 1 agonists, we carried out in vitro studies on the isolated rabbit iris-ciliary body. Sigma () recognition sites are a unique class of binding sites, heterogeneously distributed in the nervous system and in peripheral organs, that presumably serve as receptors for some unidentified endogenous ligand (Walker et al., 1990;Quiron et al., 1992). The recognition sites bind an array of structural classes of compounds including haloperidol, 1,3-di-O-tolylguanidine (DTG), and (ϩ)-benzomorphans, such as (ϩ)-pentazocine and (ϩ)-N-allylnormetazocine (Su and Junien, 1994). On the basis of biochemical and radioligand binding data, recognition sites have been classified into at least two types, 1 and 2 (Quiron et al., 1992). The 1 recognition sites display preferential affinity and stereoselectivity for (ϩ)-benzomorphans . A 1 binding protein has been cloned (Hanner et al., 1996), and its sequence shows significant similarities with sterol C 8 -C 7 isomerases from fungi.The functional role of recognition sites and the cellular mechanisms responsible for the effects produced by -site ligands have not been clearly determined, although these compounds may act as neuromodulators. Previous reports have associated -site ligands with calcium homeostasis (Brent et al., 1996;Hayashi et al., 2000;Hayashi and Su, 2001). -site ligands can influence [ 3 H]dopamine and [ 3 H]norepinephrine (NE) release from rat brain slices, acting, at least partially, presynaptically (Gonzalez-Alvear and Werling, 1995;Gonzalez and Werling, 1997).Several -site ligands influence electrically evoked contractions in the guinea pig longitudinal muscle/myenteric plexus preparation (Campbell et al., 1989). These latter findings add evidence to the theory that recognition sites participate in the regulation of autonomic functions and that -site ligands may interfere with neurotransmitter release, modulating their action on innervated tissue (Su and J...

In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (approximately 78% of cell surface receptors were lost after 2 min exposure to 1 microM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported, in SK-N-BE cells. This difference may be related to higher levels of beta-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was dependent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the plasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization. Moreover, we observed that blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.

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