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Abstract
PURPOSE. The endogenous cannabinoids N-arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are known to decrease intraocular pressure (IOP). Recently, a novel putative endogenous cannabinoid, noladin ether, was isolated in porcine and rat brains. In the present study, both the degradation of endogenous cannabinoids in ocular tissues and the effect on IOP of 2-AG and noladin ether were compared.
METHODS. The rates of enzymatic degradation for AEA, 2-AG, and noladin ether were determined in bovine cornea and iris-ciliary body homogenates. 2-AG and noladin ether were dissolved in either hydroxypropyl-β-cyclodextrin (HP-β-CD) or propylene glycol and administered unilaterally to the rabbit eye. IOPs were measured in the treated and untreated eyes. The CB1 receptor antagonist AM251 was administered topically 15 minutes before the cannabinoids to investigate whether CB1 receptors mediate the effect on IOP produced by 2-AG and noladin ether.
RESULTS. Noladin ether degraded more slowly than either 2-AG or AEA in the iris-ciliary body and cornea homogenates. The effect on IOP of 2-AG was biphasic (i.e., an initial increase in IOP followed by a reduction in the treated eye). Noladin ether decreased IOP immediately after topical administration, and no initial IOP increase was observed in the treated eye. The CB1 receptor antagonist AM251 (25 μg) blocked the effect on IOP of noladin ether but did not affect the action of 2-AG.
CONCLUSIONS. Topical administration of the novel putative endogenous cannabinoid noladin ether decreased IOP in rabbits. This IOP reduction was most probably mediated through the CB1 receptor. The effect on IOP of noladin ether differed from those of the known endogenous cannabinoids AEA and 2-AG, probably because of its more stable chemical structure.
Introduction
Evidence for an endogenous cannabinoid system was obtained in 1988 when the first cannabinoid receptor was identified in rat brain.1 Presently, the endogenous cannabinoid system has been shown to include at least two major cannabinoid receptor subtypes, CB1 and CB2,1 2 in addition to two known endogenous ligands for cannabinoid receptors, N-arachidonylethanolamide (AEA)3 and 2-arachidonylglycerol (2-AG).4 5 Recently, a novel putative endogenous CB1 receptor ligand, noladin ether, was identified in porcine6 and rat7 brains. Chemically, noladin ether is the 2-glyceryl ether of arachidonyl alcohol and structurally resembles 2-AG (Fig. 1) .
Noladin ether has been shown to exhibit several cannabimetic actions that are typical for AEA and 2-AG, such as reduced locomotor activity, catalepsy, hypothermia, and antinociception.6 It has a demonstrated binding affinity for CB1 receptors in rat brain microsomal membranes, with an inhibition constant (Ki) of 21.2 ± 0.5 nM. A considerably lower binding affinity for CB2 receptors (Ki > 3 μM) has been reported for noladin ether in CB2-transfected COS cells.6 Noladin ether exhibits agonist activities toward cells expressing CB1 and CB2 receptors, as evaluated by estimating their capacities to induce Ca2+-currents.8
As with typical neurotransmitters, AEA and 2-AG are rapidly inactivated after their biosynthesis and release. Both endogenous cannabinoids are apparently transported into cells by a common carrier-mediated uptake mechanism.9 10 In cells, AEA is enzymatically hydrolyzed to arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH),11 whereas FAAH and the yet uncharacterized lipase activities are thought to be involved in the enzymatic hydrolysis of 2-AG to arachidonic acid.9 The cellular uptake of noladin ether has recently been suggested to occur through the endocannabinoid transporter.7
In this study, the enzymatic stabilities for 2-AG, AEA, and noladin ether were compared in bovine cornea and iris-ciliary body homogenates. It has already been demonstrated that various cannabinoids affect intraocular pressure (IOP) in rabbits12 13 14 15 and humans,16 particularly in patients with glaucoma who are resistant to conventional glaucoma therapies.17 For this reason, we examined the ability of noladin ether, the more enzymatically stable endocannabinoid, to decrease IOP in rabbits and compared its effect on IOP to those of 2-AG. Finally, we examined whether the effect on IOP of 2-AG and noladin ether were mediated through CB1 receptors.
Materials and Methods
Chemicals
AEA was purchased from Deva Biotech (Hatboro, PA). 2-AG and arachidonic acid were obtained from Cayman Chemical (Ann Arbor, MI). Noladin ether (2-arachidonyl glycerol ether, HU-310) was synthesized as described earlier.6 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD, Encapsin, molecular weight [mw] = 1297.4, degree of molar substitution 0.4) was obtained from Janssen Biotech (Olen, Belgium), and the CB1 receptor antagonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) was purchased from Tocris Cookson (Bristol, UK). Propylene glycol was purchased from Oriola Oy (Espoo, Finland).
Preparation of Eye Homogenates for Degradation Study
Cornea and iris-ciliary body homogenates (20% wt/wt) were prepared as follows. Approximately 20 bovine eyes, obtained from a local abattoir (Atria Oy, Kuopio, Finland) were immediately rinsed with ice-cold 0.9% NaCl solution, and the corneas were dissected within 3 hours of death. Iris-ciliary bodies were detached from the eye as a single tissue sample. The removed eye tissues were cut into smaller pieces, weighed into separate centrifuge tubes and homogenized in tubes with four volumes of ice-cold isotonic 50 mM phosphate buffer (pH 7.4) using a homogenizer (model X-1020; Ystral, Ballrechten, Germany). The crude homogenates were centrifuged for 90 minutes at 9000g at 4°C (RC-26 Plus centrifuge; Sorvall DuPont, Newtown, CT). Pellets were discharged and supernatants were stored at −80°C until use. The protein concentrations, determined by using the Bradford method,18 were 7.1 mg/mL for cornea and 7.3 mg/mL for iris-ciliary body homogenates, respectively.
Degradation Study
The degradation rates for 2-AG, AEA, and noladin ether were determined in 10% bovine cornea and iris-ciliary body homogenates. An appropriate amount of test compound (initial concentrations were 0.7 mM for AEA and 2-AG and 0.1 mM for noladin ether) was dissolved in one volume of 50 mM isotonic phosphate buffer (pH 7.4) containing 0.1% ethanol. One volume of preheated cornea or iris-ciliary body homogenate was added, and the solution was incubated at 37°C in a water bath. At predetermined time intervals, 200-μL aliquots were withdrawn and immediately mixed with 400 μL ice-cold acetonitrile to terminate the reaction. After 5 minutes of centrifugation at 14,000 rpm (16,000g), the clear supernatant was assayed for remaining amounts of endogenous cannabinoid by HPLC.
HPLC Analysis
The analytical HPLC system (Hitachi Ltd., Tokyo, Japan) consisted of a pump (model L-7100), interface module (model L-7000), diode array detector (model L-7455; 200—400 nm, set at 211 nm) and a programmable autosampler (model L-7250). An endcapped reversed-phase column (125 mm × 4 mm, 5 μm; Purospher RP-18; VWR International, Niittyrinne, Finland), protected with an end-capped guard column (4 mm × 4 mm, 5 μm), was used for chromatographic separations.
Isocratic elutions for the hydrolysis studies in bovine iris-ciliary body and cornea homogenates were performed with a mobile phase mixture of 18% phosphate buffer (20 mM, pH 3.0) in acetonitrile at a flow rate of 1.2 mL/min. The concentrations of the eye drops were determined by a gradient method, in which the mobile phase consisted of 20 mM monobasic potassium phosphate buffer (20 mM, pH 7.0) in acetonitrile. The proportion of acetonitrile was linearly increased from 60% to 90% during a 15-minute period, maintained at a 5-minutes plateau, and subsequently returned to the initial concentration during the course of 6 minutes.
Preparation of Eye Drop Solutions
2-AG and noladin ether were dissolved either in HP-β-CD solutions or propylene glycol. The aqueous eye drop solutions were adjusted to pH 7.4 with 0.1 M sodium hydroxide solution, and the solutions were made isotonic with sodium chloride. Final eye drop concentrations in HP-β-CD were 1.25 mg/mL (12.5% HP-β-CD) and 2.5 mg/mL (25% HP-β-CD) for 2-AG and 0.625 (6.25% HP-β-CD) and 2.5 mg/mL (25% HP-β-CD) for noladin ether.
2-AG was dissolved in propylene glycol to concentrations of 0.3125, 1.25, and 5 mg/mL and noladin ether to a concentration of 5 mg/mL. A 25% HP-β-CD solution or propylene glycol was used as a control vehicle in the IOP studies. The CB1 receptor antagonist AM251 was dissolved in 45% HP-β-CD to a concentration of 0.5 mg/mL.
Animals
Normotensive pigmented Dutch belted rabbits (2.4—3.6 kg, n = 6) or New Zealand White albino rabbits (3.0—4.0 kg, n = 6), of both sexes, were used as experimental animals. The rabbits were housed singly in cages under controlled illumination (12-hour light-dark cycle, with lights-on at 7:00 A.M.) and environmental conditions (temperature, 20.0 ± 0.5°C, 55%—75% relative humidity). The rabbits were given water and food ad libitum, except during the experiments. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
IOP Measurements
For each experiment, rabbits were placed in plastic restraining boxes that were located in a quiet room. A drop (25—50 μL) of test solution was instilled unilaterally into the left eye on the upper corneoscleral limbus. During installation, the upper eyelid was gently pulled slightly away from the globe. IOP was measured by using a pneumatonometer (Digilab Modular One; Bio-Rad, Cambridge, MA) for pigmented rabbits or a handheld tonometer (Tonopen XL; Mentor, Norwell, MA) for albino rabbits. Before each measurement, 1 or 2 drops of topical anesthetic (0.04% oxybuprocaine) was applied to reduce discomfort. For every determination, at least two readings were taken from each treated (ipsilateral) and untreated (contralateral) eye, and the mean of these readings were used. IOP was measured at 1 hour before administration, then at 0, 0.5, 1, 2, 3, 4, and 5 hours after application of the eye drops. IOP at the time of administration of the eye drops (0 hour) was used as a baseline value. All IOP studies begun at 8:30 A.M., and were set up according to a nonbblinded, randomized, crossover design. At least 72 hours of washout time was allowed for each rabbit between doses. In the CB1 antagonism experiments, AM251 was administered topically 15 minutes before the topical administration of either 2-AG or noladin ether.
Statistical Analysis
The IOP results are presented as a change in IOP (% of the baseline) mean ± SE of the mean. The statistical analysis was performed on computer (SPSS ver. 11.0 for Windows; SPSS, Inc., Chicago, IL), and a two-way analysis of variance (ANOVA) for repeated measures with Bonferroni correction was used to compare treated and untreated groups. P < 0.05 was considered statistically significant.
Results
Degradation Rate of the Endocannabinoids in Eye Homogenates
The half-lives (t1/2, mean ± SD) of AEA and 2-AG in 10% bovine iris-ciliary body homogenate were 8.7 ± 3.8 minutes (n = 3) and 3.1 ± 0.9 minutes (n = 3), respectively. The corresponding t1/2 in 10% bovine cornea homogenate were 178.7 ± 21.2 minutes for AEA (n = 3) and 19.1 ± 2.6 minutes for 2-AG (n = 3), respectively. The hydrolysis of AEA and 2-AG followed pseudo first-order kinetics in the iris-ciliary body (Fig. 2) and cornea homogenates, and their main degradation products eluted at the retention time of a synthetic arachidonic acid standard (Fig. 3) .
Noladin ether degraded much more slowly in bovine iris-ciliary body (t1/2 = 430.0 ± 0.0 minute; n = 2) and cornea homogenate (t1/2 = 1128.7 ± 532.1 minutes; n = 2) than either AEA or 2-AG. Noladin ether did not degrade to arachidonic acid during the incubation time (120 minutes; Fig. 3 ). The iris-ciliary body homogenate contained low levels of arachidonic acid itself, as the HPLC chromatogram showed (Fig. 3D) . These endogenous arachidonic acid levels corresponded with the amounts found in the noladin ether samples (Fig. 3C) .
Effect on IOP
2-AG was administered topically, either in aqueous HP-β-CD or propylene glycol vehicle. The effect on IOP of 2-AG in the treated eyes of pigmented rabbits are summarized in Table 1 . Topical administration of 2-AG in the HP-β-CD vehicle induced a dose-dependent increase in IOP followed by a decrease that was, however, not statistically significant. The maximum IOP reduction (−15.4% ± 6.4%) produced by 2-AG in the HP-β-CD vehicle was obtained at 2 hours after application (Table 1) . A similar dose-dependent IOP profile was obtained for 2-AG when it was administered in a propylene glycol vehicle (Table 1) .
In contrast, noladin ether decreased IOP in the treated eyes immediately after topical administration in normotensive pigmented rabbits (Table 2) . A maximum decrease in IOP was obtained at 2 hours after the topical administration of noladin ether, and it varied between −10.3% ± 1.5% and −17.3% ± 1.6%, depending on the dose and vehicle used. In albino rabbits, noladin ether also decreased IOP in the treated eyes immediately after administration. The maximal IOP reduction (−17.7% ± 2.7%) in albino rabbits was obtained at 3 hours post-dosing at a dose of 125 μg, when given in the propylene glycol vehicle (Table 3) . The differences between the IOP profiles induced by topical 2-AG and noladin ether treatments in the treated eyes of pigmented rabbits are illustrated in Figure 4 . Neither 2-AG nor noladin ether treatments significantly affected IOP in the untreated eyes (data not shown).
Antagonism of the Effect on IOP
To investigate whether the effect on IOP of either 2-AG or noladin ether were dependent on the CB1 receptors, pigmented rabbits were pretreated with the CB1 receptor antagonist AM251. The biphasic IOP effects of 2-AG (62.5 μg in 25% HP-β-CD) were not influenced by AM251 (25 μg; Fig. 5A ). In contrast, the same dose of AM251 blocked the reduction of IOP induced by noladin ether (62.5 μg in 25% HP-β-CD; Fig. 5B ). Topical administration of AM251 without the CB1 agonist did not have any recordable effect on the IOP of pigmented rabbits. Overall, the IOP changes varied between +3.3% ± 1.7% and −1.3% ± 1.9% after the topical administration of AM251 (25 μg) alone and were not statistically significant compared with the effect of treatment with 25% HP-β-CD vehicle.
Discussion
Recently, a third putative endogenous cannabinoid, noladin ether, was isolated from porcine6 and rat brains.7 Noladin ether is chemically the 2-glyceryl ether of arachidonyl alcohol and thus differs from the previously identified endogenous cannabinoids AEA and 2-AG, which are the ethanolamide and glyceryl ester of arachidonic acid, respectively.
The present results demonstrate that noladin ether degraded at a significantly slower rate than either 2-AG or AEA in 10% bovine iris-ciliary body and cornea homogenates. The hydrolysis rates decreased in the order of 2-AG > AEA > noladin ether in both eye homogenates. These results can be explained by the fact that various esterases19 as well as FAAH-like enzyme activities20 21 are present in the eye, whereas relevant ocular enzymes for cleaving ether functional groups are probably not present. The higher hydrolysis rate for 2-AG, compared with AEA, may be because FAAH has been reported to hydrolyze 2-AG at a rate four times faster than AEA.22 In addition, these biological tissues are well known to be rich in enzymes that are capable of hydrolyzing esters. The main degradation product for AEA and 2-AG in the eye homogenates was arachidonic acid (Fig. 3) , but no arachidonic acid was formed from noladin ether during its incubation in 10% cornea and iris-ciliary body homogenates. The slow hydrolysis rate of noladin ether may raise the question of whether noladin ether is actually an endogenously produced cannabinoid receptor ligand in the ocular tissues studied. However, Fezza et al.7 have very recently suggested a shared cellular uptake for noladin ether with 2-AG and AEA, and its subsequent slow incorporation into phospholipids as an elimination mechanism in intact C6 glioma cells.
The topically administered AEA typically produces an initial IOP hypertension followed by a significant IOP reduction in the treated eye.12 23 This biphasic IOP profile for AEA is thought to be due to its rapid hydrolysis to arachidonic acid, which is subsequently biosynthesized to prostanoids that are responsible for effect on IOP.13 24 By contrast, synthetic cannabinoids, such as CP55940 and WIN55212-2, decrease IOP without an initial increase in IOP.15 25 When the effect on IOP of topically administered 2-AG and noladin ether were compared, 2-AG induced an initial IOP increase and subsequent reduction of IOP in rabbits, as reported earlier by Pate et al.14 The IOP profile of noladin ether differed from the profiles induced by other endogenous cannabinoids, because noladin ether decreased IOP immediately after topical administration without any initial elevation in IOP. On the basis of the differences in IOP profiles and degradation data (Fig. 3) , our results suggests that 2-AG is hydrolyzed, similar to AEA, to arachidonic acid in the eye, whereas noladin ether is eventually degraded to a metabolite other than arachidonic acid.
A CB1 receptor antagonist AM25126 was administered topically to study whether the IOP effects of 2-AG and noladin ether are mediated through the CB1 receptors in rabbits. AM251 blocked the IOP reduction caused by topical noladin ether but did not affect the IOP profile of 2-AG. This finding further strengthens the hypothesis that the CB1 receptors participate in the IOP effects of noladin ether. The effect on IOP of 2-AG is mediated through an alternative mechanism, most probably through its prostanoid metabolite(s). Earlier, we reported that the biphasic effect on IOP of topical AEA is not antagonized by the CB1 receptor antagonist SR141716A,25 which is also suggestive of an active prostanoid metabolite(s).
In conclusion, the present study shows for the first time that the topical administration of the putative endogenous cannabinoid, noladin ether, decreases IOP in rabbits. The present results suggest that the effects on IOP of noladin ether are mediated through the ocular CB1 receptors, whereas the effects on IOP of 2-AG are most probably mediated by its arachidonic acid metabolites. These data also demonstrate that noladin ether is the first endogenous cannabinoid identified that does not degrade to arachidonic acid after topical administration.
Source, Graphs and Figures: Comparison of the Enzymatic Stability and Intraocular Pressure Effects of 2-Arachidonylglycerol and Noladin Ether, a Novel Putative Endocannabinoid
PURPOSE. The endogenous cannabinoids N-arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are known to decrease intraocular pressure (IOP). Recently, a novel putative endogenous cannabinoid, noladin ether, was isolated in porcine and rat brains. In the present study, both the degradation of endogenous cannabinoids in ocular tissues and the effect on IOP of 2-AG and noladin ether were compared.
METHODS. The rates of enzymatic degradation for AEA, 2-AG, and noladin ether were determined in bovine cornea and iris-ciliary body homogenates. 2-AG and noladin ether were dissolved in either hydroxypropyl-β-cyclodextrin (HP-β-CD) or propylene glycol and administered unilaterally to the rabbit eye. IOPs were measured in the treated and untreated eyes. The CB1 receptor antagonist AM251 was administered topically 15 minutes before the cannabinoids to investigate whether CB1 receptors mediate the effect on IOP produced by 2-AG and noladin ether.
RESULTS. Noladin ether degraded more slowly than either 2-AG or AEA in the iris-ciliary body and cornea homogenates. The effect on IOP of 2-AG was biphasic (i.e., an initial increase in IOP followed by a reduction in the treated eye). Noladin ether decreased IOP immediately after topical administration, and no initial IOP increase was observed in the treated eye. The CB1 receptor antagonist AM251 (25 μg) blocked the effect on IOP of noladin ether but did not affect the action of 2-AG.
CONCLUSIONS. Topical administration of the novel putative endogenous cannabinoid noladin ether decreased IOP in rabbits. This IOP reduction was most probably mediated through the CB1 receptor. The effect on IOP of noladin ether differed from those of the known endogenous cannabinoids AEA and 2-AG, probably because of its more stable chemical structure.
Introduction
Evidence for an endogenous cannabinoid system was obtained in 1988 when the first cannabinoid receptor was identified in rat brain.1 Presently, the endogenous cannabinoid system has been shown to include at least two major cannabinoid receptor subtypes, CB1 and CB2,1 2 in addition to two known endogenous ligands for cannabinoid receptors, N-arachidonylethanolamide (AEA)3 and 2-arachidonylglycerol (2-AG).4 5 Recently, a novel putative endogenous CB1 receptor ligand, noladin ether, was identified in porcine6 and rat7 brains. Chemically, noladin ether is the 2-glyceryl ether of arachidonyl alcohol and structurally resembles 2-AG (Fig. 1) .
Noladin ether has been shown to exhibit several cannabimetic actions that are typical for AEA and 2-AG, such as reduced locomotor activity, catalepsy, hypothermia, and antinociception.6 It has a demonstrated binding affinity for CB1 receptors in rat brain microsomal membranes, with an inhibition constant (Ki) of 21.2 ± 0.5 nM. A considerably lower binding affinity for CB2 receptors (Ki > 3 μM) has been reported for noladin ether in CB2-transfected COS cells.6 Noladin ether exhibits agonist activities toward cells expressing CB1 and CB2 receptors, as evaluated by estimating their capacities to induce Ca2+-currents.8
As with typical neurotransmitters, AEA and 2-AG are rapidly inactivated after their biosynthesis and release. Both endogenous cannabinoids are apparently transported into cells by a common carrier-mediated uptake mechanism.9 10 In cells, AEA is enzymatically hydrolyzed to arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH),11 whereas FAAH and the yet uncharacterized lipase activities are thought to be involved in the enzymatic hydrolysis of 2-AG to arachidonic acid.9 The cellular uptake of noladin ether has recently been suggested to occur through the endocannabinoid transporter.7
In this study, the enzymatic stabilities for 2-AG, AEA, and noladin ether were compared in bovine cornea and iris-ciliary body homogenates. It has already been demonstrated that various cannabinoids affect intraocular pressure (IOP) in rabbits12 13 14 15 and humans,16 particularly in patients with glaucoma who are resistant to conventional glaucoma therapies.17 For this reason, we examined the ability of noladin ether, the more enzymatically stable endocannabinoid, to decrease IOP in rabbits and compared its effect on IOP to those of 2-AG. Finally, we examined whether the effect on IOP of 2-AG and noladin ether were mediated through CB1 receptors.
Materials and Methods
Chemicals
AEA was purchased from Deva Biotech (Hatboro, PA). 2-AG and arachidonic acid were obtained from Cayman Chemical (Ann Arbor, MI). Noladin ether (2-arachidonyl glycerol ether, HU-310) was synthesized as described earlier.6 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD, Encapsin, molecular weight [mw] = 1297.4, degree of molar substitution 0.4) was obtained from Janssen Biotech (Olen, Belgium), and the CB1 receptor antagonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) was purchased from Tocris Cookson (Bristol, UK). Propylene glycol was purchased from Oriola Oy (Espoo, Finland).
Preparation of Eye Homogenates for Degradation Study
Cornea and iris-ciliary body homogenates (20% wt/wt) were prepared as follows. Approximately 20 bovine eyes, obtained from a local abattoir (Atria Oy, Kuopio, Finland) were immediately rinsed with ice-cold 0.9% NaCl solution, and the corneas were dissected within 3 hours of death. Iris-ciliary bodies were detached from the eye as a single tissue sample. The removed eye tissues were cut into smaller pieces, weighed into separate centrifuge tubes and homogenized in tubes with four volumes of ice-cold isotonic 50 mM phosphate buffer (pH 7.4) using a homogenizer (model X-1020; Ystral, Ballrechten, Germany). The crude homogenates were centrifuged for 90 minutes at 9000g at 4°C (RC-26 Plus centrifuge; Sorvall DuPont, Newtown, CT). Pellets were discharged and supernatants were stored at −80°C until use. The protein concentrations, determined by using the Bradford method,18 were 7.1 mg/mL for cornea and 7.3 mg/mL for iris-ciliary body homogenates, respectively.
Degradation Study
The degradation rates for 2-AG, AEA, and noladin ether were determined in 10% bovine cornea and iris-ciliary body homogenates. An appropriate amount of test compound (initial concentrations were 0.7 mM for AEA and 2-AG and 0.1 mM for noladin ether) was dissolved in one volume of 50 mM isotonic phosphate buffer (pH 7.4) containing 0.1% ethanol. One volume of preheated cornea or iris-ciliary body homogenate was added, and the solution was incubated at 37°C in a water bath. At predetermined time intervals, 200-μL aliquots were withdrawn and immediately mixed with 400 μL ice-cold acetonitrile to terminate the reaction. After 5 minutes of centrifugation at 14,000 rpm (16,000g), the clear supernatant was assayed for remaining amounts of endogenous cannabinoid by HPLC.
HPLC Analysis
The analytical HPLC system (Hitachi Ltd., Tokyo, Japan) consisted of a pump (model L-7100), interface module (model L-7000), diode array detector (model L-7455; 200—400 nm, set at 211 nm) and a programmable autosampler (model L-7250). An endcapped reversed-phase column (125 mm × 4 mm, 5 μm; Purospher RP-18; VWR International, Niittyrinne, Finland), protected with an end-capped guard column (4 mm × 4 mm, 5 μm), was used for chromatographic separations.
Isocratic elutions for the hydrolysis studies in bovine iris-ciliary body and cornea homogenates were performed with a mobile phase mixture of 18% phosphate buffer (20 mM, pH 3.0) in acetonitrile at a flow rate of 1.2 mL/min. The concentrations of the eye drops were determined by a gradient method, in which the mobile phase consisted of 20 mM monobasic potassium phosphate buffer (20 mM, pH 7.0) in acetonitrile. The proportion of acetonitrile was linearly increased from 60% to 90% during a 15-minute period, maintained at a 5-minutes plateau, and subsequently returned to the initial concentration during the course of 6 minutes.
Preparation of Eye Drop Solutions
2-AG and noladin ether were dissolved either in HP-β-CD solutions or propylene glycol. The aqueous eye drop solutions were adjusted to pH 7.4 with 0.1 M sodium hydroxide solution, and the solutions were made isotonic with sodium chloride. Final eye drop concentrations in HP-β-CD were 1.25 mg/mL (12.5% HP-β-CD) and 2.5 mg/mL (25% HP-β-CD) for 2-AG and 0.625 (6.25% HP-β-CD) and 2.5 mg/mL (25% HP-β-CD) for noladin ether.
2-AG was dissolved in propylene glycol to concentrations of 0.3125, 1.25, and 5 mg/mL and noladin ether to a concentration of 5 mg/mL. A 25% HP-β-CD solution or propylene glycol was used as a control vehicle in the IOP studies. The CB1 receptor antagonist AM251 was dissolved in 45% HP-β-CD to a concentration of 0.5 mg/mL.
Animals
Normotensive pigmented Dutch belted rabbits (2.4—3.6 kg, n = 6) or New Zealand White albino rabbits (3.0—4.0 kg, n = 6), of both sexes, were used as experimental animals. The rabbits were housed singly in cages under controlled illumination (12-hour light-dark cycle, with lights-on at 7:00 A.M.) and environmental conditions (temperature, 20.0 ± 0.5°C, 55%—75% relative humidity). The rabbits were given water and food ad libitum, except during the experiments. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
IOP Measurements
For each experiment, rabbits were placed in plastic restraining boxes that were located in a quiet room. A drop (25—50 μL) of test solution was instilled unilaterally into the left eye on the upper corneoscleral limbus. During installation, the upper eyelid was gently pulled slightly away from the globe. IOP was measured by using a pneumatonometer (Digilab Modular One; Bio-Rad, Cambridge, MA) for pigmented rabbits or a handheld tonometer (Tonopen XL; Mentor, Norwell, MA) for albino rabbits. Before each measurement, 1 or 2 drops of topical anesthetic (0.04% oxybuprocaine) was applied to reduce discomfort. For every determination, at least two readings were taken from each treated (ipsilateral) and untreated (contralateral) eye, and the mean of these readings were used. IOP was measured at 1 hour before administration, then at 0, 0.5, 1, 2, 3, 4, and 5 hours after application of the eye drops. IOP at the time of administration of the eye drops (0 hour) was used as a baseline value. All IOP studies begun at 8:30 A.M., and were set up according to a nonbblinded, randomized, crossover design. At least 72 hours of washout time was allowed for each rabbit between doses. In the CB1 antagonism experiments, AM251 was administered topically 15 minutes before the topical administration of either 2-AG or noladin ether.
Statistical Analysis
The IOP results are presented as a change in IOP (% of the baseline) mean ± SE of the mean. The statistical analysis was performed on computer (SPSS ver. 11.0 for Windows; SPSS, Inc., Chicago, IL), and a two-way analysis of variance (ANOVA) for repeated measures with Bonferroni correction was used to compare treated and untreated groups. P < 0.05 was considered statistically significant.
Results
Degradation Rate of the Endocannabinoids in Eye Homogenates
The half-lives (t1/2, mean ± SD) of AEA and 2-AG in 10% bovine iris-ciliary body homogenate were 8.7 ± 3.8 minutes (n = 3) and 3.1 ± 0.9 minutes (n = 3), respectively. The corresponding t1/2 in 10% bovine cornea homogenate were 178.7 ± 21.2 minutes for AEA (n = 3) and 19.1 ± 2.6 minutes for 2-AG (n = 3), respectively. The hydrolysis of AEA and 2-AG followed pseudo first-order kinetics in the iris-ciliary body (Fig. 2) and cornea homogenates, and their main degradation products eluted at the retention time of a synthetic arachidonic acid standard (Fig. 3) .
Noladin ether degraded much more slowly in bovine iris-ciliary body (t1/2 = 430.0 ± 0.0 minute; n = 2) and cornea homogenate (t1/2 = 1128.7 ± 532.1 minutes; n = 2) than either AEA or 2-AG. Noladin ether did not degrade to arachidonic acid during the incubation time (120 minutes; Fig. 3 ). The iris-ciliary body homogenate contained low levels of arachidonic acid itself, as the HPLC chromatogram showed (Fig. 3D) . These endogenous arachidonic acid levels corresponded with the amounts found in the noladin ether samples (Fig. 3C) .
Effect on IOP
2-AG was administered topically, either in aqueous HP-β-CD or propylene glycol vehicle. The effect on IOP of 2-AG in the treated eyes of pigmented rabbits are summarized in Table 1 . Topical administration of 2-AG in the HP-β-CD vehicle induced a dose-dependent increase in IOP followed by a decrease that was, however, not statistically significant. The maximum IOP reduction (−15.4% ± 6.4%) produced by 2-AG in the HP-β-CD vehicle was obtained at 2 hours after application (Table 1) . A similar dose-dependent IOP profile was obtained for 2-AG when it was administered in a propylene glycol vehicle (Table 1) .
In contrast, noladin ether decreased IOP in the treated eyes immediately after topical administration in normotensive pigmented rabbits (Table 2) . A maximum decrease in IOP was obtained at 2 hours after the topical administration of noladin ether, and it varied between −10.3% ± 1.5% and −17.3% ± 1.6%, depending on the dose and vehicle used. In albino rabbits, noladin ether also decreased IOP in the treated eyes immediately after administration. The maximal IOP reduction (−17.7% ± 2.7%) in albino rabbits was obtained at 3 hours post-dosing at a dose of 125 μg, when given in the propylene glycol vehicle (Table 3) . The differences between the IOP profiles induced by topical 2-AG and noladin ether treatments in the treated eyes of pigmented rabbits are illustrated in Figure 4 . Neither 2-AG nor noladin ether treatments significantly affected IOP in the untreated eyes (data not shown).
Antagonism of the Effect on IOP
To investigate whether the effect on IOP of either 2-AG or noladin ether were dependent on the CB1 receptors, pigmented rabbits were pretreated with the CB1 receptor antagonist AM251. The biphasic IOP effects of 2-AG (62.5 μg in 25% HP-β-CD) were not influenced by AM251 (25 μg; Fig. 5A ). In contrast, the same dose of AM251 blocked the reduction of IOP induced by noladin ether (62.5 μg in 25% HP-β-CD; Fig. 5B ). Topical administration of AM251 without the CB1 agonist did not have any recordable effect on the IOP of pigmented rabbits. Overall, the IOP changes varied between +3.3% ± 1.7% and −1.3% ± 1.9% after the topical administration of AM251 (25 μg) alone and were not statistically significant compared with the effect of treatment with 25% HP-β-CD vehicle.
Discussion
Recently, a third putative endogenous cannabinoid, noladin ether, was isolated from porcine6 and rat brains.7 Noladin ether is chemically the 2-glyceryl ether of arachidonyl alcohol and thus differs from the previously identified endogenous cannabinoids AEA and 2-AG, which are the ethanolamide and glyceryl ester of arachidonic acid, respectively.
The present results demonstrate that noladin ether degraded at a significantly slower rate than either 2-AG or AEA in 10% bovine iris-ciliary body and cornea homogenates. The hydrolysis rates decreased in the order of 2-AG > AEA > noladin ether in both eye homogenates. These results can be explained by the fact that various esterases19 as well as FAAH-like enzyme activities20 21 are present in the eye, whereas relevant ocular enzymes for cleaving ether functional groups are probably not present. The higher hydrolysis rate for 2-AG, compared with AEA, may be because FAAH has been reported to hydrolyze 2-AG at a rate four times faster than AEA.22 In addition, these biological tissues are well known to be rich in enzymes that are capable of hydrolyzing esters. The main degradation product for AEA and 2-AG in the eye homogenates was arachidonic acid (Fig. 3) , but no arachidonic acid was formed from noladin ether during its incubation in 10% cornea and iris-ciliary body homogenates. The slow hydrolysis rate of noladin ether may raise the question of whether noladin ether is actually an endogenously produced cannabinoid receptor ligand in the ocular tissues studied. However, Fezza et al.7 have very recently suggested a shared cellular uptake for noladin ether with 2-AG and AEA, and its subsequent slow incorporation into phospholipids as an elimination mechanism in intact C6 glioma cells.
The topically administered AEA typically produces an initial IOP hypertension followed by a significant IOP reduction in the treated eye.12 23 This biphasic IOP profile for AEA is thought to be due to its rapid hydrolysis to arachidonic acid, which is subsequently biosynthesized to prostanoids that are responsible for effect on IOP.13 24 By contrast, synthetic cannabinoids, such as CP55940 and WIN55212-2, decrease IOP without an initial increase in IOP.15 25 When the effect on IOP of topically administered 2-AG and noladin ether were compared, 2-AG induced an initial IOP increase and subsequent reduction of IOP in rabbits, as reported earlier by Pate et al.14 The IOP profile of noladin ether differed from the profiles induced by other endogenous cannabinoids, because noladin ether decreased IOP immediately after topical administration without any initial elevation in IOP. On the basis of the differences in IOP profiles and degradation data (Fig. 3) , our results suggests that 2-AG is hydrolyzed, similar to AEA, to arachidonic acid in the eye, whereas noladin ether is eventually degraded to a metabolite other than arachidonic acid.
A CB1 receptor antagonist AM25126 was administered topically to study whether the IOP effects of 2-AG and noladin ether are mediated through the CB1 receptors in rabbits. AM251 blocked the IOP reduction caused by topical noladin ether but did not affect the IOP profile of 2-AG. This finding further strengthens the hypothesis that the CB1 receptors participate in the IOP effects of noladin ether. The effect on IOP of 2-AG is mediated through an alternative mechanism, most probably through its prostanoid metabolite(s). Earlier, we reported that the biphasic effect on IOP of topical AEA is not antagonized by the CB1 receptor antagonist SR141716A,25 which is also suggestive of an active prostanoid metabolite(s).
In conclusion, the present study shows for the first time that the topical administration of the putative endogenous cannabinoid, noladin ether, decreases IOP in rabbits. The present results suggest that the effects on IOP of noladin ether are mediated through the ocular CB1 receptors, whereas the effects on IOP of 2-AG are most probably mediated by its arachidonic acid metabolites. These data also demonstrate that noladin ether is the first endogenous cannabinoid identified that does not degrade to arachidonic acid after topical administration.
Source, Graphs and Figures: Comparison of the Enzymatic Stability and Intraocular Pressure Effects of 2-Arachidonylglycerol and Noladin Ether, a Novel Putative Endocannabinoid
