Ocular Hypotensive Action of Topical Flunarizine in the Rabbit: Role of 1 Recognition Sites

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 ( )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 (Ki 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. Flunarizine (IC50 5. 96 nM) and ( )-pentazocine (IC50 3. 81 nM) inhibited [ H]norepinephrine release. Moreover, flunarizine (IC50 6.34 nM) and ( )-pentazocine (IC50 27.26 nM) also antagonized isoproterenol-induced cAMP accumulation. The action of flunarizine and ( )-pentazocine was sensitive to NE-100 antagonism; however, this latter compound partially prevented their effect on [H]norepinephrine and cAMP accumulation. These findings indicate that flunarizine and ( )-pentazocine interact with ocular 1 sites and may prove effective in the control of ocular hypertension. 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, 1,3di-O-tolylguanidine (DTG), and ( )-benzomorphans, such as ( )-pentazocine and ( )-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 ( )-benzomorphans (DeHaven-Hudkins et al., 1992). A 1 binding protein has been cloned (Hanner et al., 1996), and its sequence shows significant similarities with sterol C8-C7 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 [H]dopamine and [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 Junien, 1994). Several of these studies (Brent et al., 1996; Gonzalez and Werling, 1997; Hayashi et al., 2000) proposed that 1 site-preferential ( )-benzomorphans behave as agonists. In contrast, NE-100 [N,Ndipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl] This study was supported in part by grants from the National Research Council (CNR) and from the University of Bologna (to S.S.). These authors contributed equally to this study. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.040584. ABBREVIATIONS: DTG, 1,3-di-O-tolylguanidine; NE, norepinephrine; NE-100, (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl; DuP 734, 1-(cyclopropylmethyl)-4-(2 -(4 -fluorophenyl)-2 -oxoethyl)piperidine HBr; IOP, intraocular pressure; PD, pupil diameter; S1–S4, stimulations 1 through 4; [Ca ]i, intracellular Ca 2 levels. 0022-3565/02/3033-1086–1094$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 40584/1025757 JPET 303:1086–1094, 2002 Printed in U.S.A. 1086 at A PE T Jornals on July 9, 2017 jpet.asjournals.org D ow nladed from (Tanaka et al., 1995) and DuP 734 (1-(cyclopropylmethyl)-4-(2 -(4 fluorophenyl)-2 -oxoethyl) piperidine HBr) (Gonzalez-Alvear and Werling, 1995), which have no effects by themselves but reverse the effects of 1-site agonists, are defined as antagonists. In the eye, recognition sites have been reported in bovine retinal membranes (Senda et al., 1997). In a previous study (Bucolo et al., 1999), we found distinguishable populations of 1 and 2 recognition sites in the rabbit iris-ciliary body, a structure that contains both parasympathetic and sympathetic innervation and contributes to the regulation of intraocular pressure (IOP) and pupil diameter (PD) (Nomura and Smelser, 1974). Topical ( )-pentazocine caused a doserelated reduction of IOP in ocular normotensive rabbits and in the -chymotrypsin model of ocular hypertension. This reduction was blocked by NE-100 which, by itself, had no effect on IOP. Recently, Ola et al. (2001) have confirmed, by in situ hybridization and immunohistochemistry, the occurrence of 1 sites in the mouse iris-ciliary body. Several studies have reported that topical and systemic calcium channel blockers reduce IOP in experimental animals and in humans (Netland et al., 1993; Siegner et al., 2000). This action has been observed with selective L-type calcium channel blockers such as dihydropyridines and verapamil (Segarra et al., 1993) or diltiazem (Melena et al., 1998) and of drugs such as flunarizine, a nonselective calcium blocker (Cellini et al., 1997; Osborne et al., 2002). The mechanisms underlying these effects have been explored very little. Flunarizine, a difluorinated piperazine derivative, is a mixed Land T-type calcium channel blocker (Holmes et al., 1984) and sodium channel antagonist (Pauwels et al., 1991). The complexity of its pharmacological profile is further borne out by findings suggesting that it inhibits the dopamine (DA) uptake process and binds to DA receptors, mainly of the D2 type, with an effect similar to the action of the dopaminergic antagonist haloperidol (Belforte et al., 2001). Flunarizine, as well as other DA receptor blockers, interacts with recognition sites (DeHaven-Hudkins and Fleissner, 1992), inhibits ( )-[H]pentazocine binding to 1 recognition sites (Basile et al., 1992), and has a mixed agonist-antagonist action on opioid receptors (Weizman et al., 1999). The present study was designed to investigate whether flunarizine interacted with 1 recognition sites in the rabbit iris-ciliary body by receptor binding, and to elucidate its effect on IOP in ocular normotensive albino rabbits and in the -chymotrypsin model of ocular hypertension. We extended our previous in vivo findings of the ocular effects of 1 ligands by investigating the action of ( )-pentazocine and flunarizine on electrically stimulated release of [H]NE from postganglionic sympathetic neurons and on isoproterenolinduced cAMP accumulation in isolated iris-ciliary body of albino rabbits. Materials and Methods Animals. Male New Zealand White albino rabbits (Charles River Italia, Calco, Italy) weighing 1.8 to 2.2 kg, with no signs of ocular inflammation or gross abnormality, were used. Animal procedures followed the guidelines of the Animal Care and Use Committee of the University of Bologna and conformed to the Association for Research in Vision and Ophthalmology (ARVO) resolution on the use of animals in research. Drugs and Chemicals. ( )-Pentazocine, haloperidol, DTG, flunarizine, and isoproterenol were from Sigma/RBI (Milan, Italy). Cinnarizine and desipramine hydrochloride were from ICN Pharmaceuticals (Milan, Italy). NE-100 was a kind gift from Taisho Pharmaceutical Co. (Tokyo, Japan). ( )-[H]Pentazocine, [H]DTG, and [H]NE were from Amersham Biosciences Inc. (Milan, Italy). All other compounds and reagents were purchased from Sigma-Aldrich (St. Louis, MO). Binding Assays. Membranes from the rabbit iris-ciliary body were prepared according to a procedure previously described (Bucolo et al., 1999). Rabbits were killed by i.v. injection of 0.3 ml/kg Tanax T-61 (Tanax; Aventis, Strasbourg, France), and the eyes were enucleated. The iris-ciliary body was rapidly removed, weighed, and homogenized in ice-cold 10 mM Tris-sucrose buffer (0.32 M sucrose in 10 mM Tris HCl, pH 7.4; 10 ml/g wet tissue weight) using a PotterElvejehm homogenizer. The homogenate was centrifuged at 1000g for 10 min at 4°C, and the supernatant was saved. The pellet was suspended in 2 ml/g Tris-sucrose buffer and centrifuged at 1,000g for 10 min at 4°C. The supernatants were combined and centrifuged (15 min, 31,000g, 4°C). The pellet was resuspended in 10 mM Tris HCl, pH 7.4, in a volume of 3 ml/g, and incubated for 30 min at 25°C. After recentrifugation as above, the pellet was resuspended in 10 mM Tris HCl, pH 7.4, in a final volume of 1.5 ml/g wet tissue, and aliquots were stored at 80°C until use. The protein concentration of the suspension was determined (Bucolo et al., 1999), and it corresponded to 68 3 g/100 mg of wet tissue (n 24). For 1 competition binding assays, membranes from rabbit irisciliary body (350 g of protein/assay tube) were incubated for 150 min at 37°C in 1 ml of incubation buffer (50 mM Tris HCl, pH 7.4 at 37°C) containing ( )-[H]pentazocine (3.0 nM; specific activity, 58 Ci/mmol) and 1 of 12 concentrations (10 12 to 5 10 4 M) of the unlabeled ligand under investigation (a stock solution of flunarizine 10 2 M was prepared in absolute ethanol and further diluted in incubation buffer). Nonspecific binding was defined in the presence of 10.0 M haloperidol and accounted at most for 20% of the total radioactivity retained in the filters. In these experimental conditions the apparent dissociation constant (Kd) of ( )-[ H]pentazocine was 4.6 0.6 nM (n 6) and the maximal number of binding sites (Bmax) corresponded to 212 17 fmol/mg protein (Bucolo et al., 1999). For 2 saturation studies, rabbit iris-ciliary body membranes (350 g of protein/assay tube) were incubated for 120 min at 25°C in 0.5 ml of incubation buffer (50 mM Tris HCl, pH 8.0, at 25°C) containing [H]DTG (3.0 nM; specific activity, 35 Ci/mmol), ( )-pentazocine (200 nM) to mask 1 sites (Quiron et al., 1992), and 1 of 12 concentrations (10 12 to 5 10 4 M) of the unlabeled ligand under investigation. Nonspecific binding was defined in the presence of 5.0 M DTG and accounted at most for 20% of the total radioactivity retained in the filters. In these experimental conditions the Kd of [H]DTG was 8.2 1.2 nM (n 5) and the maximal number of binding sites (Bmax) corresponded to 1120 98 fmol/mg protein (Bucolo et al., 1999). All experiments were run in duplicate, and at least three independent experiments were done. Incubations were terminated by rapid filtration (for 1-binding assays) or with 5 ml of ice-cold 10 mM Tris HCl, pH 8.0 (Tris buffer), and vacuum filtration (for 2-binding assays) through glass-fiber filters (Schleicher & Schuell, Dassel, Germany) presoaked in 0.1% polyethylenimine for at least 60 min before use. Radioactivity retained on the filters was measured by liquid scintillation spectrometry using a Beckman LS 1701 counter (after overnight incubation in scintillation cocktail), with a counting efficiency of 60%. Inhibition constants (Ki), and Hill coefficients (nH) were calculated using the LIGAND or EBDA programs (ElsevierBIOSOFT, Cambridge, UK). PD and IOP Measurements. Conscious rabbits were placed in restraint boxes to which they had been habituated, with unrestricted head and eye movements. PD (in millimeters) was measured with a Castroviejo caliper under constant light. Then, 10.0 l of 0.4% oxybuprocaine hydrochloride (Novesina; Novartis, Milan, Italy) was apOcular Actions of Flunarizine via 1 Sites 1087 at A PE T Jornals on July 9, 2017 jpet.asjournals.org D ow nladed from plied to the cornea to minimize any discomfort to the animal, and IOP (millimeters of mercury) was measured using a Tono-Pen XL tonometer (Mentor Corp., Norwell, MA), calibrated according to the manufacturer’s instructions and against a Goldmann applanation tonometer in different groups of normotensive and hypertensive rabbits (Mermoud et al., 1995); the local anesthetic had no effect on PD and IOP. Before IOP measurement, the anterior segment of each eye was macroscopically observed to check for discomfort or signs of inflammation, adopting the procedure previously described (Bucolo et al., 1999). For each IOP determination, three readings were taken on each eye, alternating the left and right eyes, and the mean was calculated. Two baseline readings were taken 30 min before and at t 0 (this was taken as baseline), and then 0.5, 1, 1.5, 2, 3, and 4 h after the instillation of eyedrops into the conjunctival sac. A stock solution of flunarizine (10 2 M) was prepared in absolute ethanol and further diluted in phosphate-buffered saline (pH 7.4; vehicle), and 50.0 l/eye was instilled. NE-100 was dissolved in phosphatebuffered saline. PD values are expressed as mean S.E.M. in millimeters; IOP values are expressed as mean S.E.M. in millimeters of mercury and as the difference from baseline. -Chymotrypsin-Induced Ocular Hypertension in Rabbit. Ocular hypertension was induced in the left eye by injection of -chymotrypsin into the posterior chamber, as described elsewhere (Sears and Sears, 1974). Briefly, a single dose of -chymotrypsin (50 Unité d’Activation Enzymatique, Pharmacopée Française; dissolved in 200 l of sterile saline) was administered using a 30-gauge needle into the posterior ocular chamber in rabbits anesthetized by an i.m. injection of 35 mg/kg ketamine (Ketalar; Parke-Davis, Milan, Italy) and 5 mg/kg xylazine HCl (Rompun 2%; Bayer AG, Leverkusen, Germany). The tip of the needle was swept across so as to distribute the enzyme evenly throughout the posterior chamber and was left in for at least 1 min before being carefully withdrawn to avoid the enzyme coming into contact with the cornea, and the external surface of the eye was washed with 10 ml of sterile saline. Ten minutes before -chymotrypsin injection and after 4, 12, and 24 h, 20 l of 0.4% oxybuprocaine hydrochloride was instilled to minimize discom-