Introduction

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The term "sigma" () is used to refer to a unique class of nonopioid, nonphencyclidine-binding sites heterogeneously distributed in the nervous system and in peripheral organs that may serve as receptors for an as-yet-unidentified endogenous ligand (Walker et al., 1990; Quiron et al., 1992; Leitner et al., 1994). The  recognition sites may bind an array of structural classes of compounds, including haloperidol, 1,3-di-o-tolylguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine [(+)-3-PPP], and (+)-benzomorphans such as (+)-pentazocine and (+)-N-allylnormetazocine [(+)-NANM] (De Costa and He, 1994). According to biochemical and radioligand-binding data,  recognition sites have been classified into at least two types, termed ₁ and ₂ (Quiron et al., 1992). Although they both bind haloperidol and DTG with high affinity, ₁ recognition sites display preferential affinity and stereoselectivity for (+)-benzomorphans (DeHaven-Hudkins et al., 1992).

A ₁-binding protein has been cloned from the guinea pig liver (Hanner et al., 1996), and its sequence shows significant similarities with sterol C₈-C₇ isomerases from fungi. This enzyme is crucial for the biosynthesis of ergosterol, which is the fungal equivalent of cholesterol; however, it remains to be confirmed whether the mammalian ₁-binding site is an enzyme involved in sterol biosynthesis (Moebius et al., 1997).

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 indicated that -site ligands may modulate agonist-stimulated phospholipase C activity in the rat brain (Bowen, 1993) and in adrenal medullary cells (Bunn et al., 1994). In rat forebrain synaptosomes, these ligands inhibit the rise of intrasynaptosomal free calcium levels induced by depolarizing agents (Brent et al., 1996). Several -site ligands may regulate N-methyl-D-aspartate-stimulated [³H]dopamine release from rat and guinea pig striatal slices (Gonzalez-Alvear and Werling, 1994, 1995; Gonzalez and Werling, 1997) or from rat hippocampal slices (Monnet et al., 1996) and affect cholinergic-dependent cognitive functions (Senda et al., 1996).

In several of the above studies (Brent et al., 1996; Monnet et al., 1996; Senda et al., 1996; Gonzalez and Werling, 1997), it was proposed that ₁-site preferential ligands such as (+)-pentazocine, (+)-NANM, and BD 737 [(+)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamine] behave as agonists. NE-100 (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl; Okuyama et al., 1993; Tanaka et al., 1995), DuP 734 [1-(cyclopropylmethyl)-4-(2'-(4"-fluorophenyl)-2'-oxoethyl)piperidine HBr], and BD 1008 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine) (Gonzalez-Alvear and Werling, 1995), which have no effects by themselves but reverse the effects of ₁-site agonists, are defined as antagonists. Interestingly, several -site ligands influence electrically evoked contractions in the isolated mouse and rat vas deferens (Campbell et al., 1987; Kennedy et al., 1990) and in the guinea pig longitudinal muscle/myenteric plexus preparation (Campbell et al., 1989). These findings add more evidence that  recognition sites may participate in the regulation of autonomic functions and that -site ligands may interfere with neurotransmitter release and/or influence their action on innervated tissue (Su and Junien, 1994).

In the eye,  recognition sites have been reported in the lachrymal gland (Schoenwald et al., 1993) and in bovine retinal membranes (Senda et al., 1997). However, it is not yet known whether  sites are present in the iris-ciliary body, which contains both parasympathetic and sympathetic innervation (Laties and Jacobowitz, 1966; Nomura and Smelser, 1974) and contributes to the regulation of intraocular pressure (IOP) and pupil diameter (PD) (Kaufman et al., 1984; Sears, 1984). The present study evaluated the presence of ₁ and ₂ recognition sites in the rabbit iris-ciliary body by receptor binding to elucidate their effects on IOP in ocular normotensive albino rabbits and in the -chymotrypsin model of ocular hypertension in the rabbit.

	Materials and Methods

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Animals. Male New Zealand White albino rabbits (Charles River, Calco, Italy) weighing 1.8 to 2.2 kg and 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. (+)-NANM, ()-NANM, (+)-pentazocine, ()-pentazocine, haloperidol, DTG, and levallorphan tartrate were from Research Biochemical International (Milan, Italy). NE-100 was a kind gift from Taisho Pharmaceutical Co. (Tokyo, Japan). 2-p-Chlorosulfophenyl-3-phenylindone was from Polysciences (Warrington, PA). [³H](+)-Pentazocine and [³H]DTG were from Amersham (Milan, Italy). All other compounds and reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Binding Assays. Membranes from the rabbit iris-ciliary body were prepared according to a procedure adopted for the guinea pig brain (Ucar et al., 1997). Male rabbits were sacrificed by i.v. injection of 0.3 ml/kg Tanax T-61 (Tanax; Hoechst AG, Frankfurt-am-Main, Germany), 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 Potter-Elvejehm 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 1000g 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 (Bradford, 1976), and it corresponded to 68 ± 3 µg/100 mg of wet tissue (n = 24).

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PD and IOP measurements. Conscious rabbits were placed in restraint boxes to which they had been habituated, with unrestricted head or eye movements. PD (in mm) was measured with a Castroviejo caliper under constant light. Then, 10 µl of 0.4% oxybuprocaine hydrochloride (Novesina; Sandoz, Milan, Italy) was applied to the cornea to minimize any discomfort to the animal, and IOP (mm Hg) was measured using a Tono-Pen XL tonometer (Mentor, Norwell, MA), calibrated according to the manufacturer's instructions; this 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 described by McDonald and Shadduck (1997). 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 at 30 min before and at t = 0 (this latter value was taken as baseline) and at 30 min and 1, 1.5, 2, 3, and 4 h after the instillation of eyedrops into the conjunctival sac. Topically administered compounds were dissolved in PBS (pH 7.4; vehicle), and 50 µl/eye was instilled. PD values are expressed as mean ± S.E.M. in mm; IOP values are expressed as mean ± S.E.M. in mm Hg 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 UAE, Pharmacopée Française; dissolved in 200 µl of sterile saline) was administered 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, Leverkusen, Germany) using a 30-gauge needle. The tip of the needle was swept across so as to distribute the enzyme evenly throughout the posterior chamber, the needle being 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 µl of 0.4% oxybuprocaine hydrochloride was instilled to minimize discomfort to the rabbit. For 7 days after -chymotrypsin injection, two chloramphenicol eyedrops (Vitamfenicolo; Allergan, Milan, Italy) were administered twice a day. In case of severe ocular inflammation (which occurred in about 5% of animals), the rabbits were not included in the study. IOP was checked after 4 weeks, and only rabbits with pressure of 26 mm Hg or more (i.e., approximately 12-15 mm Hg above the IOP in the contralateral, untreated eye) and no sign of ocular inflammation were used.

Ocular Distribution Studies. Rabbits received a topical instillation (50 µl/eye) of (+)-pentazocine (0.05% w/v) containing a trace amount of [³H](+)-pentazocine (900,000 dpm/50 µl; specific activity, 58 Ci/mmol). The animals were sacrificed by i.v. injection (in the marginal vein of the ear) of Tanax (0.3 ml/kg; Hoechst AG) 30 min after topical treatment. The cornea, iris-ciliary body, lens, and retina were separated from the remainder of the eye (the entire procedure took less then 3 min per eye), rinsed with 1 ml of ice-cold phosphate buffer (pH 7.4), and gently blotted with Kimwipes to remove residual fluid. Each tissue sample was weighed and digested in a scintillation vial at 55°C for 18 h in 1 ml of a tissue solubilizer (Solvable; Packard, Meriden, CT), and then 9 ml of a liquid scintillation cocktail was added (Filter-Count; Packard). All samples were stored in the dark for 24 h before counting in a liquid scintillation spectrometer. After being corrected for background (tissue samples from untreated rabbits were processed as above), quenching, and decay, the degradation per minute (dpm) was expressed as a percentage of the total dpm instilled per gram of wet tissue. Blocking studies were done in the same way except that 50 µl of a 0.1% solution (w/v) of NE-100 was topically instilled 10 min before the cold (+)-pentazocine mixed with the radioligand.

Statistical Analysis. Data are expressed as mean ± S.E.M. Statistical comparisons were made by ANOVA for repeated measures and posthoc Dunnett's multiple comparison test with differences of P < .05 being considered significant (GraphPAD Software, San Diego, CA).

	Results

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Binding Characteristics of  Ligands in Rabbit Iris-Ciliary Body Membranes. Saturation binding assays indicated that [³H](+)-pentazocine bound with high affinity to iris-ciliary body membranes under the experimental conditions adopted (Fig. 1). The apparent affinity of [³H](+)-pentazocine (K_d = 4.6 ± 0.6 nM; n_H = 0.92; n = 6) was similar to that detected in guinea pig brain homogenates (4.3 ± 0.8 nM; data taken from Ucar et al., 1997), whereas the B_max was lower (212 ± 17 fmol/mg protein, n = 6, versus 1998 ± 97 fmol/mg protein found in guinea pig brain homogenates; S. Spampinato, unpublished data). Saturation analysis of [³H]DTG binding, in the presence of (+)-pentazocine (200 nM) to mask ₁ sites, confirmed there were ₂-binding sites in this tissue (K_d = 8.2 ± 1.2 nM, n = 5; n_H = 0.88; B_max = 1120 ± 98 fmol/mg protein; n = 5). The affinity of [³H]DTG to iris-ciliary body membranes and to guinea pig brain membranes (K_d = 9.9 ± 0.8 nM; data from Ucar et al., 1997) was also equivalent, whereas the B_max value in iris-ciliary body membranes was lower than that in guinea pig brain membranes (B_max = 1540 ± 78 fmol/mg protein; n = 5; S. Spampinato, unpublished data). The binding of [³H](+)-pentazocine and [³H]DTG to iris-ciliary body membranes was described better by assuming an interaction of each radioligand with a single population of binding sites over the range of concentrations employed (n_H values were not significantly different from unity; p > .05).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Saturation curve (A) and Scatchard plot (B) of [3H](+)-pentazocine specific binding to rabbit iris-ciliary body membranes, investigated as described in Materials and Methods. Membranes were incubated with various concentrations of [3H](+)-pentazocine for 150 min at 37°C. Nonspecific binding was defined by addition of 10 µM haloperidol. Ligand concentrations ranged from 0.175 to 75 nM. Each point is the mean of three experiments performed in duplicate. In some cases, S.E. was smaller than the symbols.

View this table: [in this window] [in a new window]   TABLE 1 Affinities (Ki) and Hill coefficients (nH) of -site ligands for [3H](+)-pentazocine and [3H]DTG binding sites in rabbit iris-ciliary body Results from computer analysis of competition curves obtained by adding various concentrations of a competing ligand and a fixed concentration (3 nM) of [3H](+)-pentazocine or of [3H]DTG in the presence of 200 nM (+)-pentazocine. Number of experiments performed in duplicate is shown in parentheses.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Competition curves of -site ligands with [3H](+)-pentazocine for its specific binding sites in rabbit iris-ciliary body membranes. Membranes were incubated with various concentrations of -site ligands (1012 to 5 × 104 M) and 3 nM [3H](+)-pentazocine for 150 min at 37°C. Nonspecific binding was defined by addition of 10 µM haloperidol. In parentheses is the number of experiments performed in duplicate. In some cases, S.E. was smaller than the symbols.

IOP and PD. The unilateral instillation of 0.01%, 0.05%, and 0.1% solutions (w/v) of (+)-pentazocine lowered the IOP of normotensive rabbits in a dose-related manner (Fig. 3A), with a maximum drop of 2.2 mm Hg 60 min after instillation of the 0.1% solution. By 4 h, IOP had returned to baseline (Fig. 3A). The elevated IOP of the -chymotrypsinized rabbit eye was significantly lowered, in a dose-related manner, by (+)-pentazocine (0.01-0.1%) (Fig. 3B), with maximum reduction (by 14.7 mm Hg) at 60 min with the 0.1% solution; the effect lasted from 1.5 to 3 h, depending on the dose (Fig. 3B). The  receptor antagonist NE-100 [50 µl of 0.1% solution instilled 10 min before 0.1% (+)-pentazocine] and the nonselective -site ligand DTG [50 µl of 0.5% solution 10 min before 0.1% (+)-pentazocine] blocked the effect of this benzomorphan in the normotensive and hypertensive rabbit eye (Fig. 3). The vehicle alone had no effect on IOP (Fig. 3), and IOP of the contralateral eye was not affected by topical treatments (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of topical (+)-pentazocine on ipsilateral IOP and antagonism of (+)-pentazocine-induced hypotension by NE-100 and DTG. Compounds and vehicle were instilled (50 µl) in the left eye. NE-100 and DTG were administered 10 min before (+)-pentazocine. IOP responses were evaluated in ocular normotensive rabbits (A) and in rabbits pretreated with -chymotrypsin at least 4 weeks earlier (B). Each value is the mean ± S.E.M. of six or seven animals, and results are expressed as the difference in mm Hg from the pretreatment value. The average basal IOP levels were 10.5 mm Hg in normotensive and 29.7 mm Hg in hypertensive eyes. *P < .05, **P < .01 versus the corresponding vehicle-treated group (Dunnett's test after ANOVA). In some cases, S.E. was smaller than the symbols.

View this table: [in this window] [in a new window]   TABLE 2 Effect of topical ()-pentazocine, NE-100, and DTG on ipsilateral IOP in ocular normotensive rabbits Compounds were instilled (50 µl) in the left eye. Each value is mean ± S.E.M. of six or seven animals, and results are expressed as difference in millimeters of mercury (mm Hg) from pretreatment value.

View this table: [in this window] [in a new window]   TABLE 3 Effect of topical ()-pentazocine, NE-100, and DTG on ipsilateral IOP in ocular hypertensive rabbits Compounds were instilled (50 µl) in the left eye. IOP responses were evaluated in rabbits pretreated with -chymotrypsin at least 4 weeks earlier. Each value is mean ± S.E.M. of six or seven animals, and results are expressed as difference in millimeters of mercury (mm Hg) from pretreatment value.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effect of topical (+)-NANM and ()-NANM on ipsilateral IOP and antagonism of (+)-NANM-induced hypotension by NE-100. Compounds were instilled (50 µl) in the left eye. NE-100 was administered 10 min before (+)-NANM. IOP responses were evaluated in ocular normotensive rabbits (A) and in rabbits pretreated with -chymotrypsin at least 4 weeks earlier (B). Each value is the mean ± S.E.M. of six or seven animals, and results are expressed as the difference in mm Hg from the pretreatment value. The average basal IOP levels were 11.2 mm Hg in normotensive and 30.6 mm Hg in hypertensive eyes. *P < .05, **P < .01 versus the corresponding pretreatment value (Dunnett's test after ANOVA). In some cases, S.E. was smaller than the symbols.

View this table: [in this window] [in a new window]   TABLE 4 Effect of topical -site ligands on ipsilateral PD in ocular normotensive rabbits Compounds were instilled (50 µl) in the left eye. Each value is mean ± S.E.M. of six or seven animals.

Ocular Distribution. At 30 min after instillation of 0.05% (+)-pentazocine containing a trace amount of tritiated compound, there was significant penetration of radioactivity in the cornea, iris-ciliary body, lens, and retina (Fig. 5). Uptake was highest in the cornea and lowest in the lens. To examine the specificity of radioactivity uptake in different ocular tissues, the  receptor antagonist NE-100 was instilled as receptor blocker 10 min before of the radioligand. This pretreatment significantly reduced the accumulation of radioactivity in the iris-ciliary body and retina (Fig. 5). In separate experiments, the presence of (+)-pentazocine in iris-ciliary body homogenates was evaluated by reverse-phase HPLC. As shown in Fig. 6, 30 min after topical instillation of 0.05% (+)-pentazocine, a peak eluted in the same position as the standard (+)-pentazocine. The concentration of (+)-pentazocine corresponded to 4.7 ± 0.32 µg/g of wet tissue (n = 6), and considering the weight of the iris-ciliary body, 90 ± 2.3 mg/eye (n = 24), this corresponded to 1.9% of the topical dose.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Distribution of radioactivity in rabbit ocular tissues 30 min after topical instillation of a 0.05% solution of (+)-pentazocine containing a trace amount of [3H](+)-pentazocine (900,000 dpm) and blockade of radioactivity accumulation by NE-100 [administered 10 min before (+)-pentazocine]. Values are percentage of total dpm administered/g of wet tissue and are the mean ± S.E.M. of four rabbits. *P < .05 versus the corresponding (+)-pentazocine-treated group (Dunnett's test after ANOVA).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   (+)-Pentazocine in iris-ciliary body homogenates detected by reverse-phase HPLC. A, typical chromatogram obtained by UV detection from extracted iris-ciliary body homogenates of untreated rabbits that were spiked with 100 ng of (+)-pentazocine (peak 1) and 600 ng of levallorphan tartrate (internal standard; peak 2). B, typical chromatogram obtained by UV detection from extracted iris-ciliary body homogenates obtained 30 min after topical instillation (50 µl) of 0.05% (+)-pentazocine (peak 1) and then spiked with 600 ng of levallorphan tartrate (peak 2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The presence of ₁ and ₂ recognition sites was determined in rabbit iris-ciliary body homogenates by receptor-binding assay. ₁ Sites were characterized by using the dextrorotatory benzomorphan (+)-pentazocine, which is considered a preferential ligand for these sites (DeHaven-Hudkins et al., 1992). This compound bound to ₁ sites expressed in the iris-ciliary body with high affinity (K_d = 4.6 nM), comparable with the values in guinea pig brain homogenates (Tanaka et al., 1995; Ucar et al., 1997). The density of the binding sites was lower than that reported for the guinea pig. In the rabbit iris-ciliary body, ₂ recognition sites are also expressed, displaying high affinity for [³H]DTG when ₁ sites are masked with (+)-pentazocine; their maximum number was approximately five times the ₁ recognition sites. The affinity of the benzomorphans (+)-pentazocine and (+)-NANM and of the purported ₁ antagonist NE-100 (Okuyama et al., 1993; Tanaka et al., 1995) for ₁ and ₂ recognition sites was determined in competition binding experiments using [³H](+)-pentazocine and [³H]DTG, respectively. These agents showed preferential affinity for ₁ sites expressed in the rabbit iris-ciliary body; NE-100 and (+)-pentazocine had similar potency, but (+)-NANM was less potent. The present results are in agreement with data obtained by DeHaven-Hudkins et al. (1992) in guinea pig brain homogenates.

Topical (+)-pentazocine caused a significant dose-related reduction of IOP in ocular normotensive albino rabbits and lowered the elevated IOP of rabbits treated with -chymotrypsin. This model is very responsive to topical ocular hypotensive agents such as timolol (Vareilles et al., 1977) and carbonic anhydrase inhibitors (Sugrue et al., 1984) and is useful for screening compounds for ocular hypotensive activity (Sugrue, 1989). Unilateral instillation of (+)-pentazocine did not affect IOP in the contralateral eye. This indicates that the ability of (+)-pentazocine to lower IOP is, in fact, due to local action and is not the result of absorption of the drug into the circulation followed by redistribution. The experimentally elevated IOP of rabbits pretreated with -chymotrypsin was maximally decreased by 12 and 14.7 mm Hg after instillation of 0.05% and 0.1% solutions, respectively, of (+)-pentazocine (50 µl/eye). This hypotensive effect lasted 3 or 4 h, depending on the dose. Normotensive rabbits were much less susceptible to the IOP-lowering activity of topical (+)-pentazocine, a 0.1% solution reducing IOP by only 2 mm Hg at 60 min. This profile of activity is similar to that described for other ocular hypotensive drugs such as adrenergic antagonists (Lotti et al., 1984) and carbonic anhydrase inhibitors topically administered in the rabbit (Sugrue et al., 1984, 1990). The benzomorphan (+)-NANM also caused a dose-dependent decrease in IOP, but it was less potent than (+)-pentazocine: the minimum effective concentration was 0.1%. This is in agreement with binding assays in which (+)-NANM displayed less affinity than (+)-pentazocine for ₁ sites expressed in the rabbit iris-ciliary body.

Ocular hypotension elicited by (+)-pentazocine and (+)-NANM was blocked by NE-100, which, by itself, had no such effect. The present findings are consistent with those mentioned in the introduction suggesting that this compound acts as a ₁-site antagonist (Brent et al., 1996; Monnet et al., 1996; Senda et al., 1996; Gonzalez and Werling, 1997). The levorotatory isomers ()-pentazocine and ()-NANM, which have low affinity for  recognition sites (Quiron et al., 1992; De Costa and He, 1994), do not lower IOP in ocular normotensive and hypertensive rabbits. Taken together, these findings add evidence to the suggestion that (+)-pentazocine and (+)-NANM act as ₁-site agonists. Interestingly, the -site ligand DTG, binding to ₁ and ₂ sites with high affinity (Quiron et al., 1992), had no effect on IOP. These findings suggest that the ₂ recognition sites in the rabbit iris-ciliary bodies do not participate in the control of IOP and/or that DTG behaves as a ₁-site antagonist. This is borne out by the observation that topical instillation of 0.1% DTG prevented the ocular hypotensive effect elicited by (+)-pentazocine, in line with previous studies suggesting that DTG is most likely an antagonist at ₁ sites (Gonzalez-Alvear and Werling, 1995) or an inverse agonist (Monnet et al., 1996).

Unlike topical cholinergic (Kaufman et al., 1984) and adrenergic agents (Sears, 1984), ₁-site agonists did not affect PD; they also appeared to be well tolerated because they did not cause any ocular inflammatory response.

Ocular distribution studies of [³H](+)-pentazocine showed that this compound adequately penetrates the rabbit eye, with significant concentrations of radioactivity in different ocular tissues 30 min after topical administration. Uptake of the radioligand in the iris-ciliary body and retina was significantly reduced by topical pretreatment with NE-100, as expected for a receptor-specific agent. These data corroborate the existence of ₁ recognition sites in ocular tissues (Senda et al., 1997) and the feasibility of investigating the in vivo distribution profile of -site ligands using radiolabeled compounds (Musachio et al., 1994). In these experiments, the radioactivity detected in several ocular tissues may correspond to both the parent (+)-pentazocine and any metabolites. To better characterize the nature of the radioactivity in the iris-ciliary body, we measured the concentrations of (+)-pentazocine by reverse-phase HPLC 30 min after instillation of a 0.05% solution. This dose was chosen because it lowers the IOP of ocular normotensive and hypertensive rabbits. The level of (+)-pentazocine in iris-ciliary body homogenates was 4.7 µg/g of wet tissue, corresponding to 1.9% of the total amount applied. Therefore, 30 min after treatment, a significant amount of (+)-pentazocine reaches the iris-ciliary body; it is not yet metabolized and may bind to ₁ sites in this tissue because its concentration is in excess of the binding sites (as determined in binding assays, the B_max of [³H](+)-pentazocine was 212 fmol/mg of protein, or approximately 14 fmol per each iris-ciliary body, assuming that it contains 68 µg of protein).

The iris and the ciliary body are innervated by the sympathetic and parasympathetic autonomic nervous systems, which control PD, accommodation, and aqueous humor formation and drainage (Kaufman et al., 1984; Sears, 1984). Several autonomic drugs are used in ophthalmology to control ocular hypertension, which is frequent in glaucoma (Leopold and Duzman, 1986). Current pharmacological therapies for this disease aim, in fact, to lower the production of aqueous humor at the ciliary body and/or to facilitate its outflow from the angle structures (Leopold and Duzman, 1986). In the introduction, we mentioned that  recognition sites might contribute to the regulation of autonomic functions; therefore, ₁-site agonists applied topically may reduce IOP binding to ₁ sites, thus interfering with any step involved in the neurotransmission at ciliary body level. The distribution of ₁ recognition sites in the iris-ciliary body, their possible prejunctional and/or postjunctional activity, and the molecular basis for ₁-site function remain to be determined, and the effects of (+)-pentazocine and (+)-NANM on aqueous humor dynamics and on ocular blood flow remain to be clarified.

In conclusion, the present study found distinguishable populations of ₁ and ₂ recognition sites in the rabbit iris-ciliary body, an ocular structure associated with aqueous humor production and drainage. We also observed that the ₁-site agonists (+)-pentazocine and (+)-NANM, applied topically, lower IOP in ocular normotensive albino rabbits and in the -chymotrypsin model of ocular hypertension, by virtue of a local effect. These data cannot be extrapolated to humans; however, these compounds may represent a novel class of agents potentially effective in the control of ocular hypertension.