Introduction

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Dopamine is a major neurotransmitter in the central nervous system and retina. Originally, dopamine receptors were classified as two major subfamilies (D1, D2). Subsequently, a combination of approaches, including radioligand binding, and functional and molecular analyses have aided in classification of D2 dopamine receptors into D₂, D₃, and D₄ receptor subtypes (Civelli et al., 1993; Gingrich and Caron, 1993; Levant, 1997). For example, differential effects of dopamine D₂ and D₃ receptor sites have been shown in rat brain (Landwehrmeyer et al., 1993; Yamada et al., 1994). Recent studies have demonstrated the differences of absolute and relative potencies for inhibition of cAMP accumulation by agonists acting at the two (D₂ and D₃) distinct receptors (Hall and Strange, 1999). Research in the eye showed that the effects of dopamine and dopamine analogs on intraocular pressure (IOP) are complex (Potter, 1995). Because the ciliary body is the site of aqueous humor formation (Cole, 1977), this tissue represented a potential site of action for drug-induced ocular hypotension. Subsequent evidence suggests that a site of action may be dopamine receptors located on postganglionic sympathetic nerve endings of ciliary body (for reviews, see Potter et al., 1990; Potter, 1995).

This laboratory previously demonstrated that dopaminergic D₂ receptor agonists from a variety of chemical classes, e.g., ergoline, phenylethylamine, aminotetralin, and aporphine, lower IOP in animals (Potter, 1995, Potter et al., 1998) and humans (Mekki et al., 1983; Prunte and Flammer, 1995). Indirect evidence suggests that D₂ receptors are present on the terminals of postganglionic sympathetic nerves in the anterior segment of the eye. In this regard, R-()-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide (TNPA), a D₂ receptor agonist, was demonstrated to lower IOP and reduce the aqueous humor formation by interacting with D₂ receptors located on postganglionic sympathetic nerve endings (Ogidigben et al., 1993; Chu et al., 1999a). The therapeutic potential of D₂ receptor agonists as antiglaucoma drugs has been investigated, but agents with appropriate efficacy have not been identified.

The action of D₃ agonists on aqueous humor dynamics has not been thoroughly investigated. Furthermore, the mechanisms and sites of action used by D₃ agonists have not been elucidated at the tissue and/or cellular levels in the anterior segment of the eye. The purpose of this study was to investigate the IOP-lowering effects of a dopamine D₃-preferring receptor agonist 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT; Levesque et al., 1992; Damsma et al., 1993), by defining its site(s) and mechanism(s) of action. Thus, this project tested the hypothesis that D₃ receptors modulate ocular hydrodynamics by examining 1) dose-related responses of 7-OH-DPAT on IOP in normal rabbits; 2) 7-OH-DPAT-induced IOP-lowering effects in sympathetically denervated rabbits and in the presence of D₃ and/or D₂ receptors antagonists; 3) aqueous humor flow rates in normal rabbits under control conditions and after acute 7-OH-DPAT treatment; and (4) localization of D₃ receptors in the ciliary processes of normal and surgically sympathectomized rabbits by immunohistochemistry.

	Materials and Methods

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Animals. New Zealand White rabbits (2-4 kg) of either sex were used to study IOP and aqueous humor flow rate. One group of rabbits (n = 4) was subjected to unilateral surgical sympathectomy. After sympathetic denervation, rabbits were allowed to recover for 2 weeks before further experimentation. Animal care and treatment were in accordance with the resolution in the use of animals for research established by National Institutes of Health and the Association for Research in Vision and Ophthalmology.

Sympathectomy. Surgical sympathectomy was performed in rabbits anesthetized with pentobarbital (30 mg/kg i. v.). The cervical sympathetic trunk and superior cervical ganglion were isolated surgically, and the successful isolation was confirmed by stimulating the nerve trunk while observing the dilation of the pupil. The right superior cervical ganglion and two millimeters of the postganglionic sympathetic nerve trunk were removed surgically (sympathectomized eyes); the superior cervical ganglion remained intact in the left eye to serve as a control. The sympathectomized eyes were tested 2 weeks after surgery for the lack of response to 1% hydroxyamphetamine, an indirect-acting sympathomimetic amine; normally innervated eyes responded with a brisk mydriasis, whereas sympathectomized eyes did not respond. Thus, 2 weeks after sympathectomy, IOP experiments were performed and samples of ciliary body tissues were removed for immunochemical analysis.

Chemicals Maleate, 7-OH-DPAT, U-99194A, spiperone hydrochloride, and raclopride tartrate were obtained from Research Biochemicals/Sigma (Natick, MA). UH232 maleate was purchased from Tocris Cookson Inc. (Ballwin, MO). Rabbit anti-human dopamine D₃ receptor antibody was purchased from Alpha Diagnostic International (San Antonio, TX). All other chemicals used in this study were obtained from Sigma (St. Louis, MO). Because UH232 is relatively insoluble in water, it was dissolved in an aqueous solution of 2-hydroxy-propyl--cyclodextrin (Chu et al., 1999b).

IOP Measurement. IOP (mm Hg) was measured with a calibrated pneumatonometer (model 30; Mentor Co., Norwell, MA). Tetracaine (0.1%; 25 µl), a local anesthetic agent, was applied to each cornea before the IOP measurements. Two baseline readings were taken at 0.5 and 0 h before topical administration of 7-OH-DPAT and postdrug determinations of ocular pressures were made at 0.5, 1, 2, 3, 4, 5, and 6 h. At the end of each series of measurements, stability of the tonometry was confirmed with the verifier supplied by the manufacturer. In other experiments to confirm that the receptors mediating 7-OH-DPAT's effect on IOP were D₃ receptors, raclopride, U-99194A, or UH232 bilateral pretreatment was performed topically and bilaterally, 30 min before 7-OH-DPAT administration.

Aqueous Humor Inflow. Aqueous humor flow rates were measured in normal rabbit eyes with fluorescein dilution as quantified by a Fluorotron Master (Ocumetrics, Palo Alto, CA). The uniform stromal depot method, described previously by Yablonski et al. (1978), was used to load fluorescein into the eyes. The general method of fluorometric measurement and calculation of aqueous humor flow rate have been described previously by Brubaker (1989) and Ogidigben et al. (1994). Basal control recordings of flow rate were established in the first week. The following week, rabbit eyes of the same group were treated bilaterally with 7-OH-DPAT, and flow rates in both eyes were determined. Fluorometric recordings started at 1, 0 h before topical administration with 7-OH-DPAT, and subsequent recordings were made at 1-h intervals for 4 h.

Immunohistochemistry. Ciliary processes were dissected from normal and sympathetically denervated rabbit eyes. The tissues from left (normal) eye were used as control for the right (sympathectomized) eye. The ciliary processes from both eyes were embedded into OCT compound (Sakura, Inc., Torrance, CA), frozen with liquid nitrogen, and kept at 20°C until processing. Subsequently, the sections were cut with a cryostat (10 µM; Leica 3050) and collected on poly(L-lysine)-coated glass slide. Ciliary process sections in the slide were fixed with 4% paraformaldehyde in PBS, pH 7.0, for 30 min, then permeabilized by 0.5% Triton X-100 with BSA (1%) in PBS. The sections of ciliary processes were incubated with primary antibodies to D₃ receptors (10 µg/ml) overnight at 4°C. After exposure to receptor antibodies, sections were counterstained with fluorescein isothiocyanate (FITC) for 2 to 4 h at room temperature. The intensity of the FITC was observed under an Olympus IX70 fluorescent microscope. The images of normal and sympathectomized ciliary processes were examined, compared, and stored in a computer.

Statistical Analysis. In dose-response studies, baseline IOP readings from vehicle-treated eyes were compared with drug-treated eyes at the same time periods. In experiments involving pretreatment with antagonists, the IOP responses to 7-OH-DPAT alone and to antagonists plus 7-OH-DPAT were compared against the responses to antagonists alone. The statistical comparisons of drug-induced changes were made with an ANOVA for multiple group comparisons followed by Student-Newman-Keuls test (Instat program; GraphPad, San Diego, CA). The level of significance was chosen as P < .05. For data presented in graphs from in vivo experiments, mean ± S.E were calculated from multiple determinations. Dose-response curves were analyzed by a computerized nonlinear curve-fitting program (Sigmaplot; Jandel Scientific Inc., Corte Madera, CA) as previously described (Crosson, 1995).

	Results

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IOP in Normal Rabbits. To characterize the ocular action of topically administered 7-OH-DPAT, the dose (25, 75, and 750 µg)-dependent effects on IOP were investigated. As shown in Fig. 1, unilateral topical application of 7-OH-DPAT caused dose-related ocular hypotension in ipsilateral (treated, Fig. 1A) and contralateral (Fig. 1B) eyes. The IOP-lowering effect of 7-OH-DPAT (75 µg) lasted ~4 h, and then IOP gradually returned to baseline level. At 750 µg, the maximum IOP-lowering effect (9 mm Hg) occurred at 1 h and persisted for 5 to 6 h. Furthermore, the peak reduction in IOP induced by 7-OH-DPAT (250 and 1000 µg) occurred at 1 h was 7 and 8.5 mm Hg, respectively. The effective dose of half-maximal reduction (ED₅₀) of IOP response produced by 7-OH-DPAT was 41 µg. The maximal peak reduction of IOP, R_max, was 9 mm Hg. Thus, the maximum IOP-lowering effect was achieved in the 750- to 1000-µg range. Finally, there were no changes in pupil diameter of normal rabbits at the doses tested.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Unilateral topical application of 7-OH-DPAT (25, 75, and 750 µg; n = 4) on IOP of normal, ipsilateral (treated, A) and contralateral (B) eyes of rabbits. The average of control IOP was 22.5 ± 1.5 mm Hg. *P < .05, significantly different from the control.

IOP in Sympathetically Denervated Rabbits. To determine possible neuronal involvement in IOP responses, the mid-range dose of 7-OH-DPAT was investigated in unilaterally sympathectomized rabbits. After topical application, 7-OH-DPAT (75 µg) lowered the IOP bilaterally in normal rabbits by 6 mm Hg at 2 h. In contrast, the reduction of IOP at 2 and 3 h, when the same dose was administered to sympathectomized rabbit eyes the response was less than that of normal rabbits (Fig. 2). These data suggest that suppression of sympathetic neuronal function by 7-OH-DPAT contributed to the lowering of IOP.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Topical administration of 7-OH-DPAT (75 µg; n = 4) on IOP of normal and sympathetically denervated (SX) eyes of rabbits. *P < .05, significantly different from the control.

Antagonism of 7-OH-DPAT-Induced Ocular Hypotension. To confirm the involvement of a D₃ receptor mechanism, experiments were performed in which pretreatment with dopamine receptor antagonists was used to investigate the ocular hypotension induced by 7-OH-DPAT. In the initial study, the D₂/D₃ receptor antagonist raclopride (Rogoz and Dziedzicka-Wasylewska, 1999) was used to discern the involvement of D₃ receptors in the anterior segment of eye. Results showed that raclopride (750 µg) antagonized the 7-OH-DPAT (75 µg)-induced IOP-lowering effects (Fig. 3). To determine the contribution of D₃ receptors, D₃ receptor antagonists UH232 and U-99194A (Audinot et al., 1998; Yamada et al., 1999) were used subsequently. Experiments were conducted in which bilateral pretreatment with UH232 (250 µg) or U-99194A (1000 µg) was followed by a subsequent challenge with 7-OH-DPAT (75 µg). Neither UH232 nor U-99194A, given alone, produced any significant change in IOP. However, the depression of IOP by 7-OH-DPAT was antagonized significantly by UH232 (Fig. 4). As shown in Fig. 5, pretreatment of U-99194A completely antagonized the IOP-lowering effect induced by 7-OH-DPAT. In this laboratory, pretreatment with spiperone (250 µg), a D₂ receptor antagonist, has been shown to inhibit effectively TNPA (75 µg)-induced ocular hypotension in rabbits (our unpublished data). However, at dose of 250 µg, spiperone did not antagonize the 7-OH-DPAT (75 µg)-induced ocular hypotension (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Antagonism of 7-OH-DPAT-induced (75 µg; n = 4) ocular hypotension in the presence of raclopride (750 µg) in normal, ipsilateral (treated, A) and contralateral (B) eyes of rabbits. *P < .05, significantly different from raclopride-treated eyes.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Reduced effect of 7-OH-DPAT (75 µg; n = 4) on IOP in the presence of UH232 (250 µg) in normal, ipsilateral (treated, A) and contralateral (B) eyes of rabbits. *P < .05, significantly different from UH232-treated eyes.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Antagonism of 7-OH-DPAT's (75 µg; n = 4) effects on IOP in the presence of U-99194A (1000 µg) of normal, ipsilateral (treated, A) and contralateral (B) eyes of rabbits. *P < .05, significantly different from U-99194A-treated eyes.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Inhibition of 7-OH-DPAT's (75 µg; n = 4) effects on IOP in the presence of spiperone (250 µg) in normal, ipsilateral (treated, A) and contralateral (B) eyes of rabbits. *P < .05, significantly different from spiperone-treated eyes.

Suppression of Aqueous Inflow by 7-OH-DPAT. This phase of this study was conducted to determine possible changes in the rate of aqueous humor inflow evoked by topically administered 7-OH-DPAT in normal rabbit eyes. Previous studies reported that the aqueous humor flow rate was reduced after topical application of the D₂ dopamine receptor agonist TNPA (Ogidigben et al., 1993). Therefore, it was considered reasonable to suggest that D₃ receptor agonists might lower IOP, in part, by suppressing the aqueous flow rate. The alteration in rate of aqueous humor inflow by 7-OH-DPAT was measured in normal rabbit eyes with fluorescein dilution as quantified by one-dimensional scanning fluorophotometry (Fluorotron Master). Topical application of 7-OH-DPAT (750 µg) caused a significant reduction of aqueous humor inflow rate at 1 to 4 h (Fig. 7). This result could be correlated in time with the decline in IOP.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Suppression of aqueous humor flow rate (microliters per minute) by 7-OH-DPAT (750 µg; n = 4) when topically administered to rabbit eyes. 7-OH-DPAT was administered at 0 time (see arrow). *P < .05, significantly different from the basal rate.

Localization of D₃ Receptors by Immunohistochemistry. Based on the reduction of 7-OH-DPAT-induced ocular hypotension after surgical sympathectomy, it was hypothesized that one of the principal sites of action of 7-OH-DPAT could be on postganglionic sympathetic nerves in the ciliary body. To determine the localization of D₃ receptors within the ciliary body, an immunochemical approach was used. In eyes with intact postganglionic sympathetic neurons, Fig. 8A shows an image of a ciliary process with strongly fluorescent signals within the bilayered ciliary epithelium (D₃ receptors-FITC). In contrast, Fig. 8B shows a significantly weaker fluorescent signal within the bilayer of the ciliary epithelium in a sympathetically denervated eye.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Immunofluorescence of D3 dopamine receptors in ciliary process in normal (A) and sympathetically denervated (B) rabbit eyes with D3 dopamine receptor antibody-FITC.

	Discussion

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Previously, we reported that D₂ receptor agonists (e.g., TNPA and lisuride) induced dose-related ocular hypotension in rabbits (Ogidigben et al., 1993; Potter et al., 1998). Because the D₃ receptor subtype belongs to the D2 subfamily, we sought to test the hypothesis that the topical application of D₃-preferring receptor agonists can depress IOP. Indeed, topical, unilateral application of 7-OH-DPAT caused dose-related, bilateral IOP-lowering effects. Similarly, past studies showed that topical application of agonists for dopamine (D₂) receptors, adrenergic ₂/imidazoline receptors and serotonin (5HT_1A) receptors evoked a bilateral reduction of IOP. In same cases, the contralateral IOP response was postulated to be mediated, in part, through an effect in central nervous system (Ogidigben et al., 1993, 1994; Campbell and Potter, 1995; Chu et al., 1999b). Based on the topical administration of radiolabeled 8-hydroxy-2-dipropylaminotetralin, the redistribution of the drug through the systemic circulation to the contralateral eyes was minimal (Chidlow et al., 1999). This finding argues against redistribution of drug as being the reason for the contralateral ocular hypotensive response. In the current study, ocular hypotension in the contralateral eyes evoked by unilateral administration of 7-OH-DPAT is postulated to be a centrally mediated response.

Moreover, another supposition to be examined was whether the IOP-lowering effects of 7-OH-DPAT would persist after loss of postganglionic sympathetic nerves by surgical removal of the superior cervical ganglion. Based on previous studies, basal IOP of surgically denervated rabbit eyes was 2 to 3 mm Hg lower than those of normal rabbits (Gregory et al., 1985; Chu et al., 1999a). Past studies have shown that norepinephrine levels in aqueous humor declined considerably as a result of sympathetic neuronal degeneration in the ciliary body and iris (Sears et al., 1966; Chu et al., 1999a). Moreover, previous studies revealed that ocular hypotensive responses for certain neuronally active agonists are markedly attenuated in sympathectomized eyes (Ogidigben et al., 1993, 1994; Chu et al., 1996, 1999a). Current experimental results confirmed that 7-OH-DPAT-induced ocular hypotension was diminished substantially in the sympathetically denervated rabbit eyes. Thus, it is suggested that suppression of activity of the peripheral sympathetic nervous system plays a role in the regulation of aqueous humor dynamics by 7-OH-DPAT; however, these results did not localize the site of action definitively. Furthermore, the direct involvement of D₃ receptors in the ocular hypotensive activity of 7-OH-DPAT in the rabbit eye had not been delineated at this point.

In this study, it was presumed that activation of D₃ receptors by 7-OH-DPAT in the anterior segment of the eye would result in ocular hypotension. In experiments testing the ocular hydrodynamic effects of 7-OH-DPAT, the antagonists U-99194A, UH232, raclopride, and spiperone were used because of their relative selectivity for D₃ and/or D₂ receptors. Experimental data showed the greater effectiveness of U-99194A's, UH232's, and raclopride's antagonistic action on the ocular hypotensive action of the relatively selective D₃ receptor agonist 7-OH-DPAT. Because spiperone induced no antagonism of the ocular hypotensive effect of 7-OH-DPAT, D₂ receptors are not considered to be a principal site of action of 7-OH-DPAT in the anterior segment of the eye. Alternatively, the data resulting from the use of U-99194A suggest that 7-OH-DPAT lowered IOP, in part, by activation of D₃ receptors in the anterior segment of eye.

Previous studies showed that TNPA, a D₂ dopamine receptor agonist, reduced the aqueous humor flow rate in rabbits (Ogidigben et al., 1993). To date, there have been limited studies examining the hydrodynamic mechanism(s) by which the D₃-preferring receptor agonist 7-OH-DPAT lowers IOP of normal rabbit eyes. The experimental outcomes described in this report demonstrate that the IOP-lowering effect of 7-OH-DPAT also might be due to inhibition of aqueous humor formation. Because the ciliary body is the site of aqueous humor formation (Cole, 1977), it is suggested that ciliary body represented a potential site of action for 7-OH-DPAT-induced ocular hypotension. However, these data do not preclude an ability of 7-OH-DPAT to lower IOP by other mechanisms based on a modest, residual response in sympathectomized eyes.

Because 7-OH-DPAT was hypothesized to lower IOP in normal rabbits, in part, by suppressing sympathetic neuronal function, it was considered important to determine whether the potential site of action of this drug was on postganglionic sympathetic nerve endings in the anterior segment. To confirm the presumed site of action, it was essential to localize D₃ receptors on the postganglionic sympathetic nerve endings of ciliary body. Recently, dopamine (D₂) receptors in ciliary body were identified by immunohistochemistry (Chu et al., 1999a). To date, the influence of sympathectomy on the integrity of D₃ receptors has not been investigated in the ciliary body of rabbit eyes by immunohistochemical techniques. To confirm the involvement of D₃ receptors, sites in ciliary process were identified by immunofluorescent staining with antibodies to D₃ receptors. Results from these immunolocalization experiments demonstrated that D₃ receptors were present on postganglionic sympathetic nerves in the ciliary processes of normal rabbit. In contrast, minimally detectable fluorescence was observed in the ciliary processes of sympathectomized rabbit eyes. This constitutes the first report of dopamine (D₃) receptors on postganglionic sympathetic nerves within the ciliary processes of normal rabbits identified by immunohistochemistry.

Based on the data presented, it is concluded that 1) the IOP-lowering effect caused by 7-OH-DPAT was due, in part, to the suppression of aqueous humor flow; 2) immunohistochemical data confirms the present of D₃ receptors in the ciliary body of normal rabbits and is consistent with the presumed site of the IOP-lowering effects by D₃ receptor agonist 7-OH-DPAT; 3) the lack of 7-OH-DPAT-induced ocular hypotension in sympathetically denervated rabbit eyes and the decreased fluorescence in the ciliary processes from sympathetically denervated eyes provided additional evidence of a site of action of 7-OH-DPAT on postganglionic sympathetic nerves; and 4) antagonism of 7-OH-DPAT-induced ocular hypotension by the D₃ receptor antagonists UH232 and U-99194A, coupled with the immunohistochemical data, suggest that one of the primary sites of D₃ receptor-mediated action is located on postganglionic sympathetic nerve endings in the ciliary body.