236 Br J Ophthalmol 1999;83:236–240

Human retina contains polyamine sensitive Br J Ophthalmol: first published as 10.1136/bjo.83.2.236 on 1 February 1999. Downloaded from [3H]-ifenprodil binding sites: implications for neuroprotection?

N A Sharif, S X Xu

Abstract pears to be primarily involved in causing retinal Aims—This study characterised the phar- ganglion cell death.9–12 macology of [3H]-ifenprodil binding to the Optic neuropathies and ischaemic borne polyamine binding sites (PBS) on the retinopathies, including glaucoma, are multi- N-methyl-D-aspartate (NMDA) receptor faceted diseases of the eye which can cause channel complex on human retinas. These blindness.13 Elevated intraocular pressure data were correlated with the known neu- (IOP) is a major risk factor in the aetiology of roprotective eVects of ifenprodil and glaucoma.13 However, increased intravitreal eliprodil. glutamate levels have been recently observed in Methods—Specific binding of [3H]- glaucoma patients and in experimentally in- ifenprodil (under sigma site blockade) was duced glaucoma in monkeys.14 Hence, elevated investigated using human retinal homoge- vitreal glutamate levels coupled with retinal nates and radioligand binding techniques. ischaemia5111415 and ocular hypertension may Scatchard and competition analyses were be responsible for retinal ganglion cell loss and utilised to define the pharmacology of the mechanical damage to the optic nerve head, [3H]-ifenprodil binding sites. eventually leading to visual field deficits and 13 Results—Specific binding of [3H]- blindness. Therapeutic intervention to pre- ifenprodil comprised 73% (SEM 3%) of serve vision in the long term may therefore not total and reflected interaction with two soley rely on IOP lowering drugs13 but also on drugs which specifically block the neurotoxic aYnity sites (Kds = 0.39 and 4.3 µM) of dif- eVects of excessive amounts of glutamate at the ferent densities (Bmax = 14.4 and 105 pmol/ mg protein) (n = 5). The rank order of retinal ganglion cell level.91516 aYnity of compounds competing for [3H]- The NMDA receptor is composed of a ifenprodil binding to the high aYnity PBS number of diVerent subunits forming the ion was: ifenprodil > eliprodil > arcaine > channel, with additional modulatory sites on spermine > diaminodecane > spermidine the receptor ionophore complex including the polyamine binding sites and strychnine insensi-

> putrescine >> MK-801 (n = 3–7). http://bjo.bmj.com/ However, [3H]-ifenprodil binding was tive glycine sites.217Eliprodil (SL82.0175) and minimally inhibited by glutamate, ifenprodil are potent neuroprotectants18–20 be- NMDA, and kainate. lieved to elicit their protective actions by block- 18 19 Conclusion—These studies have shown, ing the polyamine binding site and/or the for the first time, the presence of specific NR2B subunit on the NMDA receptor channel [3H]-ifenprodil binding sites in the human complex17 and thus preventing the sequelae of retina with pharmacological characteris- neurotoxic events mentioned above. Since tics of PBS associated with the NMDA eliprodil has recently been shown to protect rat on September 25, 2021 by guest. Protected copyright. receptor ionophore complex. The neuro- retinal ganglion cells from NMDA induced protective eVects of eliprodil and ifen- toxicity12 in vitro and to preserve retinal prodil may, in part, be mediated via these architecture and function in rats and rabbits [3H]-ifenprodil labelled sites. subjected to excitotoxic and ischaemic insults 21 (Br J Ophthalmol 1999;83:236–240) in vivo, we decided to investigate whether spe- cific polyamine binding sites existed in human retinas which may help explain the neuropro- The excitatory amino acids, glutamate and tective eVects of eliprodil and ifenprodil men- aspartate, are important neurotransmitters in tioned above. Therefore, the aims of the present Molecular the retina and the rest of the central nervous studies were to investigate the presence of spe- Pharmacology Unit, system (CNS).1–3 However, excessive accumu- cific [3H]-ifenprodil binding sites on washed Alcon Laboratories, lation of these amino acids in the extracellular homogenates of human retinas and to charac- Inc, Fort Worth, Texas, space can lead to neuronal death via excitotox- USA terise these sites pharmacologically, relating this N A Sharif icity mediated calcium overloading, production information to the NMDA receptor and its SXXu of free radicals, and activation of intracellular modulatory sites to functions in the retina. To proteases.45Ischaemic insults in the retina can the best of our knowledge, this represents the Correspondence to: also excerbate the situation by enhancing gluta- first such study to address these topics. N A Sharif, PhD, Molecular Pharmacology Unit (R2–19), mate release and thus increasing its neurotoxic 6 Alcon Laboratories, Inc, potential. The toxic eVects of glutamate in the Materials and methods 78 6201 South Freeway, Fort retina have been known for a long time. PREPARATION OF RETINAL HOMOGENATES Worth, TX 76134, USA. Although several subtypes of the glutamate Human retinas were obtained from human 45 Accepted for publication receptor can mediate neurotoxicity, the donor cadaver eyes within 8–18 hours of death 1 September 1998 N-methyl-D-aspartate (NMDA) subtype ap- and immediately frozen in liquid nitrogen until Retinal [3H]-ifenprodil binding 237 Br J Ophthalmol: first published as 10.1136/bjo.83.2.236 on 1 February 1999. Downloaded from

10 100

80

8

60 H]-ifenprodil binding 3

40 ifenprodil 6 eliprodil arcaine diaminodecane

Bound free ( × 100; pmol/mg/nM) 20 spermine spermidine % Max specific [ 4 putrescine MK-801 0 0 10 20 30 40 50 60 70 —9 —8 —7 —6 —5 —4 —3 Bound (pmol/mg protein) log [Drug] (M) Figure 1 Scatchard analysis of specific [3H]-ifenprodil Figure 2 Competition curves for various compounds binding to human retinal homogenates. Experiments were 3 conducted in the presence of 3 µM DTG and 10 µM displacing [ H]-ifenprodil binding from polyamine binding GBR12909 to block the radioligand from binding to sigma sites on homogenates of human retinas. The assays were binding sites. Plots shown are from one sample experiment conducted in the presence of 3 µM DTG and 10 µM (average of triplicates per point) for illustrative purposes, GBR12909 to block the radioligand (2 nM final) from but composite data from five independent experiments are binding to sigma binding sites. The dpm bound in the shown in the Results section. presence of each competing ligand concentration were converted to an overall percentage of maximum specific [3H]-ifenprodil binding. Data shown are from one used for the radioligand binding experiments. experiment (average of triplicates per point) for illustrative None of the donors had any documented ocu- purposes, but composite data, in terms of aYnity values

lar diseases and the mean ages of the donors (Kis), from 3–7 experiments are shown in the Results were 80 (SEM 2) years (32 donors). The section. pooled frozen thawed retinal tissue (usually MATERIALS from six pairs of eyes at a time) was gently Human cadaver donor eyes were obtained from homogenised in 50 volumes of ice cold 5 mM local eye banks within 8–18 hours after death TRIS. HCl (pH 7.4) using a Polytron tissue and transported in ice cold Dexol or Optisol disrupter (setting 5–7 seconds) in a laminar 3 ° corneal preservation medium. [ H]-ifenprodil flow hood and centrifuged at 4 Cat30000g (68.1 Ci/mmol) was purchased from Amer- for 30 minutes. The resultant pellets were

sham Corp (Arlington Heights, IL, USA), http://bjo.bmj.com/ resuspended by homogenisation in 20 volumes while unlabelled drugs including ifenprodil of fresh 5 mM TRIS HCl buVer, recentrifuged ((±))-(R*,S*)-a-(4-hydroxyphenyl)-á-methyl-4- as above and then resuspended in more fresh (phenylmethyl)-1-piperidine-ethanol-(R,R)-2,3 buVer for the binding assays. -dihydroxy-butanedioate(2:2) (salt), hemihy- drate), were purchased from Research Bio- RECEPTOR BINDING ASSAYS chemical, Int (Natick, MA, USA). However, The ligand binding assays were performed as ± 22 eliprodil HCl (( )-a-(4-chlorophenyl)-4[(4- previously described. The retinal homoge- fluorophenyl)methyl]-1-piperidine-ethanol) was on September 25, 2021 by guest. Protected copyright. nates (0.2 mg protein final), containing 3 µM a generous gift from Synthelabo Recherche DTG (1,3-di(2-tolyl)guanidine, HCl) and 10 (Bagneux, France). All other standard chemi- µM GBR12909 (final assay concentrations) to 3 cals and reagents were purchased from Sigma block [ H]-ifenprodil binding to sigma binding Chemical Company (St Louis, MO, USA). sites22–24 were incubated with 2–2.5 nM [3H]- ifenprodil in the absence or presence of 1 mM spermine in a total volume of 500 µl for 30 DATA ANALYSIS The original data (dpm bound) were analysed minutes at 20°C to define total and non- using a non-linear, iterative curve fitting specific binding, respectively. Since DTG and computer program25 26 incorporating a logistic GBR12909 have aYnities of 40–60 nM for the function. The competition data for ifenprodil sigma sites,23 the concentrations employed in v [3H]-ifenprodil were processed using the the current studies were expected to block all “EBDA” suite of computer programs27 to per- the sigma binding sites in the retinal homoge- form Scatchard analysis and thus derive the nates based on the fractional receptor occu- apparent receptor aYnity (K ) and apparent pancy principle. The incubations were termi- d density (B ) parameters. Data are presented nated by rapid vacuum filtration over Wallac max as mean (SEM) from several experiments. glass fibre GF/B filters previously soaked in 0.3% polyethyleneimine using 12 ml of ice cold 50 mM TRIS.HCl (pH 7.4 at 4°C). Filter Results bound radioactivity was determined on a â [3H]-Ifenprodil (2.0–2.5 nM final) (in the scintillation counter and the data analysed presence of 3 µM DTG and 10 µM using non-linear, iterative curve fitting compu- GBR12909) binding to human retinal ter programs (see below). homogenates (0.2 mg protein/assay tube) 238 Sharif, Xu

retinal preparations with diVerent aYnities Br J Ophthalmol: first published as 10.1136/bjo.83.2.236 on 1 February 1999. Downloaded from A = Ifenprodil (Fig 2; Table 1). The rank order of compound 80 B = Eliprodil aYnities was: ifenprodil > eliprodil > arcaine > C = Glutamate spermine > diaminodecane > spermidine > D = Kainate putrescine >> MK-801 (n = 3–7). Again, pre- E = NMDA liminary studies on rat and rabbit retinas 60 F = AMPA yielded similar data (for example, see Table 1). In view of the apparent biphasic nature of the competition curves for ifenprodil competing for [3H]-ifenprodil binding in the retinal H]-ifenprodil binding 3 40 preparations, the data were fitted to the two site model and were reasonably well resolved into two components. The following apparent aYnity data for the two sites were obtained: 20 IC50high = 0.1 (0.02) µM (45% (5%) of sites) and IC = 12.1 (4.4) µM (54% (5%) of

% Inhibition of [ 50low sites). Ligands known to be potent and selective agonists at the glutamate receptor 0 subtype binding sites, such as kainate, NMDA, A B C DEF AMPA ((±)-á-amino-3-hydroxy-5-methyl- Figure 3 EVect of various glutamate receptor binding site isoxazole-4-propionic acid hydrobromide) and ligands and polyamine antagonists, ifenprodil and eliprodil, glutamate itself, exhibited low aYnity (<20% on [3H]-ifenprodil binding to human retinal homogenates. inhibition at 10 µM final concentration; n = 3) Compounds were tested at 10 µM and the data shown are 3 mean from > 3 experiments; vertical lines are SEM. for [ H]-ifenprodil binding to human retinal homogenates under sigma site blockade condi- Table 1 AYnities of polyamines and polyamine tions (Fig 3). antagonists for [3H]ifenprodil binding to human retinal homogenates Discussion Apparent aYnity in Polyamine binding sites represent novel modu- human retina Pseudo-Hill Compound (K ;µM) coeYcient (nH) latory sites on the heteromeric NMDA recep- i tor channel complex which regulate ionic Ifenprodil 0.7 (0.1) 0.6 (0.1)* fluxes across neuronal cell membranes.17 19 Eliprodil 6.8 (0.7) 1.0 (0.1) 3 125 Arcaine 20.2 (4.7) 0.8 (0.1) [ H] and [ I]-ifenprodil have been used to Spermine 27.6 (2.6) 1.0 (0.1) selectively label these sites in various CNS Diaminodecane 51.2 (15.3) 0.8 (0.2) 22 28 29 Spermidine 81.2 (15.9) 0.8 (0.1) tissues. In the current studies we dupli- Putrescine >560 — cated these assay conditions and have success- fully demonstrated, for the first time, the pres- Data are mean (SEM) from 3–7 independent experiments. [3H]ifenprodil binding was conducted in the presence of 3 µM ence of specific polyamine binding sites in the DTG and 10 µM GBR12909 to block sigma binding sites. human retinal tissues. Using Scatchard and *nH significantly <1, and dissection of the biphasic competition competition analyses, [3H]-ifenprodil binding http://bjo.bmj.com/ curves yielded the data presented in the Results section. Preliminary studies with rat and rabbit retinas yielded the in the retinal preparations could be resolved following estimated aYnities: ifenprodil = 0.5–0.9 µM, eliprodil into two apparent binding sites of submicro- = 0.9–1.1 µM, spermine = 47–67 µM, spermidine = >100 µM molar and micromolar aYnities. The aYnity and putrescine = >650 µM. variables and the pharmacological properties of the high aYnity sites in the human retina comprised 2870 (SEM 139) dpm total binding closely matched those previously reported for 22 and 790 (99) dpm non-specific binding (yield- rat cerebral cortical synaptic membranes, rat on September 25, 2021 by guest. Protected copyright. ing 73% (3%) specific binding; 2080 dpm spe- neonate cerebellar membranes,29 and in rat cific), respectively. Scatchard analyses of com- brain sections studied by autoradiography.28 petition data identified two apparent classes of The neuroprotective and anti-ischaemic ef- diVerent aYnity sites of the specific [3H]- fects of the polyamine antagonists, eliprodil ifenprodil binding to polyamine binding sites and ifenprodil, in a variety of cells/tissues in in the human retina (Fig 1). Estimated equilib- vitro and in many animal models in vivo are 19 rium variables (dissociation constant [Kd] and now well documented (see Scatton et al for

apparent density of binding sites [Bmax]) for review). Additionally, these drugs have been [3H]-ifenprodil binding to the retinal homoge- shown to potently and eYcaciously antagonise

nates were: dissociation constant (Kd1) = 390 a variety of NMDA induced responses in vitro

(80) nM and apparent density of sites (Bmax1)= such as preventing NMDA induced neurotox- 12 14.4 (2.9) pmol/mg protein; (Kd2) = 4300 icity and ionic fluxes in retinal ganglion cells 30–32 (900) nM and Bmax2 = 105 (19) pmol/mg and brain cells and blocking calcium entry protein (n = 5 experiments). Preliminary stud- into neurons,33 34 blocking transmitter release 35 ies on rat and rabbit retinas yielded Kd1 = 300– from striatum and blocking NMDA induced

500 nM and Bmax1 = 2–4 pmol/mg protein; Kd2 electrophysiological responses in rat retinal 12 33 = 2–4 µM and Bmax2 = 2–11 pmol/ mg protein ganglion cells and other neurons. In vivo, (data not shown). eliprodil has been shown to prevent NMDA Eliprodil, ifenprodil, arcaine, diamino- induced retinal ischaemia and excitotoxicity in decane, and polyamines such as spermine, the rabbit leading to preservation of the spermidine and putrescine, and other com- electroretinogram (ERG).21 Ifenprodil and pounds of interest, concentration dependently eliprodil antagonised the majority of the inhibited [3H]-ifenprodil binding in the human above mentioned functional physiological and Retinal [3H]-ifenprodil binding 239

pathological responses with potencies in the polyamine modulatory sites are also primarily Br J Ophthalmol: first published as 10.1136/bjo.83.2.236 on 1 February 1999. Downloaded from submicromolar to micromolar range, thus present on retinal ganglion and amacrine 42 matching the aYnity values (Kd and Ki values) cells. obtained for [3H]-ifenprodil binding in the It is concluded that human retinas possess human retinal preparations in our current specific [3H]-ifenprodil binding sites, at rela- studies. Furthermore, the aYnity values for the tively high concentrations, which exhibit phar- polyamine antagonists/NR2B antagonists macological properties akin to the polyamine (eliprodil, ifenprodil, arcaine, and diamino- binding sites previously defined in numerous decane) and polyamine agonists (spermine, CNS tissues and cells. These specific 3 spermidine, and putrescine) obtained here in polyamine sensitive [ H]-ifenprodil binding the human retina (Table 1) also closely resem- sites may be involved in mediating some or all bled those previously reported for rat brain, of the neuroprotective eVects of ifenprodil and cerebral cortex, and cerebellum.22 28 29 Like- eliprodil discussed above. The data presented in this communication suggest that since wise, recent preliminary studies on rat and rab- [3H]-ifenprodil binding sites are present in the bit retinas yielded similar drug aYnities for human retinas that polyamine antagonists like displacing [3H]-ifenprodil binding from human eliprodil and ifenprodil may also exhibit retinas. The relatively low aYnity of MK 801 3 neuroprotective properties in the human retina in competing for [ H]-ifenprodil binding to in vitro and perhaps in vivo. Studies to retinal homogenates is consistent with the fact elucidate such activities of these compounds that MK-801 binds to the NMDA receptor 225 are therefore warranted and remain to be con- channel as opposed to interacting with the ducted. modulatory polyamine sites on the receptor channel complex as do the polyamine agonists 19 36 The authors thank Dr M Kapin (Alcon) and Drs B Scatton and and antagonists. The reason for putrescine H Schoemaker (Synthelabo Recherche, Bagneux, France) for showing a lower aYnity than spermine or sper- providing helpful comments. The human donors and their families are gratefully acknowledged for making the studies on midine is the fact that it is a precursor of the the human retinas possible. latter compounds.36 37 As expected, glutamate receptor site selective compounds (glutamate, 1 Sharif NA. Multiple synaptic receptors for neuroactive NMDA, AMPA, kainate) exhibited minimal amino acid transmitters—new vistas. Int Rev Neurobiol 3 1985; 26:85–150. aYnities for the specific [ H]-ifenprodil bind- 2 Collingridge GL, Lester RA. Excitatory amino acid ing sites in human retinal homogenates (Fig 3), receptors in the vertebrate nervous system. Pharmacol Rev 1989;40:143–210. thus confirming similar observations for rat 3 Zhang D, Sucher NJ , Lipton SA. Co-expression of AMPA/ CNS tissues29 and providing further evidence kainate receptor-operated channels with high and low Ca2+ permeability in single rat retinal ganglion cells. Neuroscience for the polyamine sites being diVerent from the 1995;67:177–88. receptor binding sites and/or the receptor 4 Choi DW. Excitotoxic cell death. J Neurobiol 1992;23:1261– 76. ionophore sites associated with the NMDA 5 Lipton SA, Rosenberg PA. Excitatory amino acids as a final receptor channel complex in the retina. common pathway for neurologic disorders. N Engl J Med 1996;330:613–22. The NMDA receptor is an heteromeric 6 Louzada-Junior P, Dias JJ, Santos WF, et al. 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Hypoxic and excitotoxic on September 25, 2021 by guest. Protected copyright. damage to cultured rat retinal ganglion cell. Exp Eye Res the polyamine binding sites. Our current aYn- 1996;63:105–12. ity data for these drugs in the retinal homoge- 11 Siliprandi R, Canella R, Carmignoto G, et al. N-methyl-D- 3 aspartate-induced neurotoxicity in the adult rat retina. Vis nates suggest that [ H]-ifenprodil may also be Neurosci 1992;8:567–73. labelling the NR1A/NR2B subunits of the 12 Pang I-H, Wexler EM, Walters RJ, et al. Protective eVects of eliprodil in retinal and neuronal cells and tissues. Soc Neu- NMDA receptor in the retinal tissue in rosci 1996;Abstract no 5072. addition to labelling the polyamine binding 13 Quigley HA. Open-angle glaucoma. N Engl J Med 1993;328:1097–106. sites. We currently do not have any evidence for 14 Dryer EB, Zurakowski D, Schumer RA, et al. 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