Introduction

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NO is the oxidation product of one of the guanidino nitrogens of L-Arg as a result of catalysis mediated by the two different forms of NOS: constitutive NOS and iNOS (Moncada et al., 1991). iNOS is expressed only on stimulation with endotoxin or combinations of inflammatory cytokines, including interferon-, tumor necrosis factor- and interleukin-1 (Goureau et al., 1994; Liversidge et al., 1994; Zidek and Frankova, 1995). In the eye, NO plays a cytostatic or cytotoxic role in the conjunctiva (Meijer et al., 1996), the retinal pigmented epithelium (Goureau et al., 1994; Liversidge et al., 1994) and the anterior uveal tract (Mandai et al., 1994) during inflammation.

Recently, an endogenous NOS inhibitor, N^G,N^G-dimethyl-L-arginine, has been found to exist at 0.2 to 0.5 µM in the bovine ciliary muscle to regulate NO synthesis (Azuma et al., 1997). In addition to N-iminoethyl-L-ornithine and N^G-amino-L-arginine (Moncada et al., 1991), NOS inhibitors such as L-NA, L-NAME and L-MMA have been reported to lower NO production in human (Goureau et al., 1994) and rat (Liversidge et al., 1994) retinal pigmented epithelial cells and the rat anterior uveal tract (Mandai et al., 1994). Thus, Goureau et al. (1994) reported that cytokine-induced nitrite formation in cultured human retinal pigmented epithelial cells was reduced by 84%, 88% and 78% in the presence of 0.1 mM L-NA, L-NAME and L-MMA, respectively. Mandai et al. (1994) also observed that L-NA administered intraperitoneally (20 mg/rat) inhibited iNOS activity by 80% in endotoxin-induced uveitis. However, there has been no report on the topical ocular delivery of NOS inhibitors for possible treatment of intraocular inflammatory conditions. Given its juxtaposition to the uveal tract, the conjunctiva may offer an attractive pathway for topically applied NOS inhibitors to gain intraocular access (Ahmed and Patton, 1987).

In a previous study (Hosoya et al., 1997), we provided evidence for the involvement of system B^0,+, a Na⁺-dependent transporter of neutral and basic amino acids (Winkle et al., 1990), in the transport of L-Arg in the pigmented rabbit conjunctiva. We also showed that the NOS inhibitors L-NA, L-NAME and L-MMA inhibited L-Arg transport to varying degrees. The purpose of the present study was to determine whether system B^0,+ was indeed involved in the conjunctival transport of NOS inhibitors. L-NA, one of the more potent NOS inhibitors (Moncada et al., 1991; Goureau et al., 1994), was chosen as a model compound because it was the only NOS inhibitors commercially available in radiolabeled form.

	Materials and Methods

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Animals. Male Dutch-belted pigmented rabbits, weighing 2.5 to 3.0 kg, were purchased from American Rabbitry (Los Angeles, CA). The investigations using rabbits described in this report conformed to the Guiding Principles on the Care and Use of Animals (DHEW Publication, NIH 80-23).

Chemicals. L-NA, L-Lys, L-Glu, L-Leu and ouabain were obtained from Sigma Chemical (St. Louis, MO). D-NA was obtained from Research Biochemicals (Natick, MA). N^G-Nitro-L-[2,3,4,5-³H]arginine HCl (³H-L-NA; specific activity, 57 Ci/mmol) and ²²NaCl (666 mCi/mg Na⁺) were purchased from Amersham (Downers Grove, IL).

Buffer solutions. Unless otherwise indicated, all experiments were conducted in bicarbonated Ringer's solution maintained at 37°C and pH 7.4 under 95% air/5% CO₂. Bicarbonated Ringer's solution contained 111.5 mM NaCl, 4.8 mM KCl, 29.2 mM NaHCO₃, 0.75 mM NaH₂PO₄, 1.04 mM CaCl₂·2H₂O, 0.74 mM MgCl₂·6H₂O and 5 mM D-glucose. Na⁺-free Ringer's solution was prepared by equimolar replacement of NaCl, NaH₂PO₄ and NaHCO₃ with choline chloride, KH₂PO₄ and choline bicarbonate, respectively. Ringer's solutions containing 8.8, 17.6, 35.3, 52.9, 70.5 and 105.8 mM Na⁺ were prepared by mixing appropriate proportions of Na⁺-free Ringer's solution with normal Ringer's solution.

Tissue preparation. We previously reported detailed methods for preparing the excised pigmented rabbit conjunctiva for Ussing chamber studies (Kompella et al., 1993). Briefly, rabbits were killed with an injection of 85 mg/kg sodium pentobarbital solution into a marginal ear vein. The entire eyeball was removed from the orbit, taking care not to damage the conjunctival epithelium. Within 15 min of surgery, the excised conjunctiva was mounted onto a tissue adapter with a circular aperture of 1.0 cm². The adapter-tissue assembly was then placed in a modified Ussing chamber maintained at 36 ± 1°C by a circulating water bath. The bathing solution of the tissue was bubbled with 5% CO₂ and 95% air to maintain the pH at 7.4 and to provide adequate agitation of the solution.

Measurement of bioelectric parameters. All experiments were performed under short-circuit condition with the use of an automatic voltage-clamp device (558C-5; Bioengineering Department, University of Iowa, Iowa city, IA). The potential difference (PD) was measured with two matched calomel electrodes. Two polyethylene (PE 90) bridges (containing 4% agar in 3 M KCl), whose tips were located near the center of tissue surfaces, were used to connect the reservoir fluid electrically to the electrode wells. The electrical output of matched calomel electrodes was amplified by the voltage-clamp unit. Direct current flowing across the tissue was measured with a pair of matched Ag/AgCl electrodes with conducting agar bridges, whose tips were positioned away from the tissue surfaces at the far ends of two reservoirs. The I_sc flowing in the bath-tissue-bath circuit was monitored and recorded on one channel of a strip-chart recorder (Kipp and Zonen, Delft, Netherlands). At 60-sec intervals, a 2-mV voltage pulse (V) was imposed for 3 sec across the short-circuited tissue to estimate the TEER as a surface area normalized ratio of applied pulse voltage to observed deflection in resultant current (I) flowing on top of I_sc, TEER = (V/I)A, where A is the nominal surface area of the Ussing chamber opening (1 cm²). Before each experiment, the solution resistance (<100 ·cm²) was compensated for by the automatic voltage-clamp unit (Kompella et al., 1993). The PD, I_sc and TEER of the conjunctiva were monitored continually. The baseline PD was 15.4 ± 0.4 mV (tear-side negative), the I_sc was 12.5 ± 0.3 µA/cm² and the TEER was 1283 ± 37 ·cm² (n = 173). These values were comparable to those reported previously (Kompella et al., 1993; Hosoya et al., 1997).

Measurement of L-NA and Na⁺ fluxes. Unidirectional L-NA or Na⁺ fluxes across the conjunctiva were determined separately using ³H-L-NA or ²²Na, respectively, at 1 µCi/ml. Then, 500-µl samples were collected from the receiver side at 0.5, 1.0, 1.5, 2.0 and 3.0 hr for assay of radioactivity in a liquid scintillation counter (LS 1801; Beckman, Fullerton, CA). The I_sc and TEER were monitored periodically to ascertain tissue viability and integrity.

Data analysis. Unidirectional fluxes (J) for L-NA and Na⁺ were estimated from the steady-state rate of radioactivity appearance in the receiver fluid. The P_app of L-NA was estimated from the steady-state slope of a plot showing cumulative amount of radioactivity vs. time according to the equation: P_app = (dQ/dt)/(A·C₀), where dQ/dt is the steady-state transport rate (mol/sec), A is the nominal surface area of the Ussing chamber opening and C₀ is the initial radioactivity concentration (mol/ml) in the donor fluid.

	Results

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L-NA transport characteristics. L-NA, when applied to the mucosal side of the conjunctiva at 37°C, increased the I_sc, as shown in figure 1. The increase in I_sc (I_sc) was 0.15 ± 0.04 µA/cm² at 0.02 mM, increasing to 1.73 ± 0.1 µA/cm² at 1 mM L-NA. Lineweaver-Burk plot of I_sc vs. [L-NA] yielded an apparent K_m value of 0.21 mM L-NA and a maximal I_sc (I_scmax) value of 1.68 µA/cm² (r² = .99). By contrast, no I_sc change was observed with serosally added L-NA (1 mM) or mucosally added L-NA (0.02-1 mM) at 4°C (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Saturable increase in Isc (Isc) as a function of L-NA concentration. Each point represents mean ± S.E.M. (n = 3-15).

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View larger version (18K): [in this window] [in a new window]   Fig. 2.   Time course of L-NA transport across the pigmented rabbit conjunctiva at 0.005 mM (), 0.01 mM (), 0.1 mM (), 0.5 mM (), 1.0 mM () and 2.0 mM (). Each point represents mean ± S.E.M. (n = 3-7).

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View larger version (15K): [in this window] [in a new window]   Fig. 3.   L-NA flux across the pigmented rabbit conjunctiva as a function of L-NA concentration at 37°C () and 4°C (). Each point represents mean ± S.E.M. (n = 3-7).

Stoichiometry of Na⁺-coupled L-NA transport. ³H-L-NA flux also saturated with Na⁺ concentration, reaching a value of 19.1 ± 0.1 pmol/(cm²·min) at 17.6 mM and 59.4 ± 5.7 pmol/(cm²·min) at 141 mM (fig. 4). The K_m value was 58.3 mM for Na⁺ and the J_max value was 86.2 pmol/(cm²·min) according to Lineweaver-Burk analysis. Hill analysis yielded a coupling ratio of 0.98 (r² = 0.97), suggesting 1:1 coupling between Na⁺ and L-NA.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   L-NA flux across the pigmented rabbit conjunctiva as a function of Na+ concentration at 37°C. Each point represents mean ± S.E.M. (n = 3).

Inhibition of Na⁺-coupled L-NA transport by select amino acids. The basic amino acids, L-Arg and L-Lys, and the neutral amino acid, L-Leu, significantly reduced ms L-NA transport (P < .05), whereas D-NA and the acidic amino acid, L-Glu, exerted no such effect (table 2). Eadie-Scatchard analysis of ms L-NA transport in the presence of 0.05 mM L-Arg showed competitive inhibition of L-NA transport by L-Arg with a K_i value of 0.034 mM (fig. 5).

View this table: [in this window] [in a new window]   TABLE 2 Effect of a few select amino acids on the permeability coefficient of [3H]L-NA in the pigmented rabbit conjunctiva [3H]L-NA (1 µCi/ml) transport across the pigmented rabbit conjunctiva was measured in the presence of 1 mM of each amino acid. [3H]L-NA transport in the presence of each inhibitor was expressed as percentage of control Papp of 15.0 ± 1.0 × 106 cm/sec (n = 4). Results represent mean ± S.E.M. (n = 4). Numbers in parentheses represent percentage of control.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Eadie-Scatchard analysis for L-Arg inhibition of saturable Na+-dependent L-NA transport. Each point represents mean ± S.E.M. (n = 3-7). , Control; , with 0.05 mM L-Arg.

Effect of L-NA on Na⁺ absorption. Na⁺ transport measured across the conjunctiva in the absence and presence of L-NA is shown in table 3. Each tissue served as its own control for the L-NA effect. L-NA at 1 mM significantly increased the ms Na⁺ flux (J_Na^ms) (P < .05) while not affecting the sm Na⁺ flux (J_Na^sm). The net Na⁺ flux (J_Na^net) before and after adding 1 mM L-NA was 0.14 and 0.23 µEq/(cm²·hr), respectively, leading to a net stimulation of 0.09 µEq/(cm²·hr) in Na⁺ absorption. Statistically, J_Na^net and I_sc are not different (P > .05).

View this table: [in this window] [in a new window]   TABLE 3 Na+ fluxes (J) across the rabbit conjunctiva in the absence and presence of 1 mM L-NA added mucosally Each value represents mean ± S.E.M. Numbers in parentheses are the number of tissues used. Net Na+ flux (JNanet) equals Na+ flux in the ms direction (JNams) minus Na+ flux in the sm direction (JNasm). JNanet equals JNanet observed at 1 mM L-NA minus JNanet observed under control conditions. All experiments were conducted in the presence of 1 µCi/ml 22Na in either mucosal (ms) or serosal (sm) fluid under short-circuit condition. JIsc,eq was calculated from the observed Isc of 1.66 ± 0.18 µA/cm2 [1 µA/cm2 = 0.0373 µEq/(cm2 · hr)] induced by 1 mM L-NA. JNanet is not statistically different from JIsc,eq (P > .05).

	Discussion

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The present study demonstrated that as was the case for L-Arg, L-NA transport across the pigmented rabbit conjunctiva was carrier-mediated, probably via system B^0,+. Aside from the kinetic characteristics of carrier-mediated transport (as shown in table 1 and fig. 3), L-NA transport was reduced to 10% of its base-line value by basic amino acids (e.g., L-Arg and L-Lys) and neutral amino acids (e.g., L-Leu) but was not affected by acidic amino acids (e.g., L-Glu) (table 2). L-Arg transport across the conjunctiva exhibited very similar inhibitory patterns (Hosoya et al., 1997). It should, therefore, not be surprising that L-Arg competitively inhibited L-NA transport with an apparent K_i value of 0.034 mM (fig. 5), which is comparable in value to the K_m value of 0.07 mM for high affinity L-Arg transport. As was the case for L-Arg transport (Hosoya et al., 1997), the stoichiometry between Na⁺ and L-NA was 1:1 (fig. 4). The sharing of a common transporter between L-Arg and L-NAME was also observed in human hepatic plasma membrane vesicles (Inoue et al., 1993).

Nevertheless, there exist some subtle differences between L-NA and L-Arg transport. First, whereas L-Arg transport is associated with both a high and a low affinity process, with a corresponding K_m value of 0.07 mM and 5.90 mM (Hosoya et al., 1997), L-NA transport is characterized by a single kinetic process with a K_m value of 0.35 mM. In addition, the K_i value for the inhibition of L-NA transport by L-Arg, 0.034 mM, is 10 times lower than the K_m value of 0.35 mM for L-NA transport. Second, the pigmented rabbit conjunctiva has a 4 times lower capacity for transporting L-NA than L-Arg, as a comparison between the J_max of L-NA [290 pmol/(cm²·min)] and that of L-Arg in the low affinity process [1248 pmol/(cm²·min)] would reveal. Third, the I_sc (1.73 ± 0.01 µA/cm²) and J_Na^net [0.09 µEq/(cm²·hr)] in the presence of 1 mM L-NA are 30% smaller than those exhibited by L-Arg transport (Hosoya et al., 1997).

Although the basal NO level in the conjunctiva is negligible (Hosoya et al., 1997), NO is known to be elevated in the conjunctiva (Meijer et al., 1996), retinal pigment epithelium (Goureau et al., 1994; Liversidge et al., 1994) and uveal tract (Mandai et al., 1994) as a result of iNOS activity. When L-NA was administered intraperitoneally (20 mg/rat) for endotoxin-induced uveitis, iNOS activity was inhibited by 90% (Mandai et al., 1994). Similarly, intraperitoneally administered L-NA (25-50 mg/kg) 1 hr before the induction of ischemic injury to the retina of the rat prevented the increase in thickness of the inner retinal and plexiform layers (Geyer et al., 1995). Finally, intravenously administered L-NA (200 mg/kg) completely blocked the inflammatory response in the rabbit eye induced by a 2-min infrared irradiation to the iris (Wang and Håkanson, 1995). Collectively, these findings suggest that L-NA may potentially be useful for alleviating ocular inflammation. The stage is therefore set for exploring the feasibility of delivering this and other NOS inhibitors topically at perhaps a much lower dose than the intravenous and intraperitoneal doses noted above.

It is conceivable that a L-NA concentration exceeding 1 µM may be achieved in the vitreous fluid from a 10-µl topical dose of 1 mM L-NA solution (containing 2.2 µg of drug). The necessary conditions are (1) the entire bulbar conjunctiva of 3.3 cm² (Hosoya and Lee, 1997) is accessible to drug delivery, (2) the conjunctiva rather than the sclera is rate-limiting in drug transport, (3) drug clearance by the vasculature in the conjunctiva and sclera is negligible, (4) the drug permeability as measured in vitro is comparable to that in vivo and (5) a minimum of 5 min in residence time for the instilled dose is allowed. Given that the maximal transconjunctival flux of L-NA is 290 pmol/(cm²·min) (fig. 3), the total amount of drug in the vitreous will be 4.8 nmol [= flux (290 pmol/(cm²·min) × area (3.3 cm²) × residence time (5 min)]. Moreover, because 99% of the vitreous body (4 ml) is water (Berman, 1991), L-NA concentration of 1.2 µM (0.26 µg/ml) can be achieved in the vitreous. This concentration is close to the IC₅₀ value of 1.4 µM for L-NA against purified rat brain NOS (Pfeiffer et al., 1996).

In conclusion, the findings in the present study are consistent with the possible involvement of system B^0,+ in the transport of L-NA and perhaps other NOS inhibitors across the pigmented rabbit conjunctiva. Compared with L-Arg, L-NA shows less affinity (and a smaller maximal saturable flux) toward the conjunctival L-Arg transporter (B^0,+). On the basis of the observed P_app and on the expectation that a delivery system targeting the bulbar conjunctiva will be available, it may be possible to deliver a therapeutic amount of L-NA to the uveal tract from the topical route. Further in vivo experimentation would be required to determine the extent to which such a desirable therapeutic goal can be achieved.