Effect of Brimonidine on Rabbit Trabecular Meshwork Hyaluronidase Activity

Jorge Benozzi, Carolina O. Jaliffa, Francisco Firpo Lacoste, Diego Weinberg Llomovatte, Marı´a I. Keller Sarmiento, and Ruth E. Rosenstein

PURPOSE. To study the presence of hyaluronidase activity in the rabbit trabecular meshwork and its regulation by brimonidine.

METHODS. A spectrophotometric assay that consists of the assessment of N-acetylhexosamine groups released from hyaluronic acid was used to examine hyaluronidase activity. Cyclic adenosine monophosphate (cAMP) levels were assessed by radioimmunoassay.

RESULTS. Hyaluronidase activity was detected in the rabbit trabecular meshwork. Its optimal activity was in the acid range of pH 3.8. Brimonidine significantly increased trabecular hyaluronidase– specific activity and decreased cAMP accumulation. Yohimbine significantly inhibited the effect of brimonidine on both hyaluronidase activity and cAMP accumulation.

CONCLUSIONS. The finding of endogenous hyaluronidase activity in rabbit trabecular meshwork supports the hypothesis that this tissue can metabolize its own glycosaminoglycan (GAG) products. The present results suggest, however, that the hypotensive effect of brimonidine could be medi- ated, at least in part, by its ability to increase GAG catabolism, probably through a cAMP- independent mechanism. (Invest Ophthalmol Vis Sci. 2000;41:2268–2272)

he primary site of aqueous humor outflow resistance dase the resistance of the filtering angle dropped to approxi- resides within the trabecular meshwork and possibly mately one half the initial value, much attention has been Twithin the deep portion of the corneoscleral meshwork devoted to the hyaluronidase-sensitive mucopolysaccharides in and/or the amorphous juxtacanalicular basement membrane the outflow apparatus. Although testicular hyaluronidase has near Schlemm’s canal. The trabecular meshwork is composed been reported to increase outflow facility in guinea pigs8 and of sheets of trabecular beams that contain lamellae made of dogs,9 the evidence suggests that it has little effect on human extracellular matrix materials, which comprise a significant eyes.10 Further investigations showed that Streptomyces hyal- portion of this tissue and probably of the outflow barrier. uronidase is considerably more effective than the testicular Among the materials of the trabecular extracellular matrix, enzyme in the rabbit eye.11 However, no increase in outflow glycosaminoglycan (GAG) profile (i.e., hyaluronic acid [HA], facility was found with acute Streptomyces hyaluronidase treat- keratan sulfate, heparan sulfate, and hybrid dermatan sulfate– ment in monkeys.12 Intense histochemical staining observed in chondroitin sulfate) has been identified in rabbits,1 monkeys,2 the various layers of human trabecular meshwork suggests that and human eyes.2 Extensive evidence indicates that GAGs of a substantial amount of HA is present in the outflow path- the trabecular extracellular matrix regulate outflow through way,13 and a quantitative analysis has indicated that it is the connective tissue and modulate outflow resistance. Moreover, most abundant GAG of the human trabecular meshwork.2 in the trabecular meshwork of patients with primary open- Although the biosynthesis of acid mucopolysaccharides in tra- angle glaucoma, several electron microscopic, histologic, and becular cells has been conclusively demonstrated,14 the mech- immunologic studies have noted excessive accumulation of anism of its degradation remains incompletely understood. extracellular matrix materials.3–5 An abnormal accumulation of Although the modulation of extracellular matrix materials acid mucopolysaccharides in the anterior chamber angle was in the trabecular meshwork by substances such as ascorbic described in steroid-induced ocular hypertension.6 acid15 and glucocorticoids6 has been demonstrated, the effect Since Barany and Scotchbrook7 reported that after treat- of medication on the trabecular meshwork biochemistry is an ment of excised cattle eyes with bovine testicular hyaluroni- open question; it is not known what if any influence drug therapy may have on the expression of GAGs in this tissue. Brimonidine is a relatively new, highly selective, and potent

From the Laboratorio de Neuroquı´mica Retiniana y Oftalmologı´a ␣2-adrenoreceptor agonist that has been shown to decrease Experimental, Departamento de Bioquı´mica Humana, Facultad de Me- intraocular pressure (IOP), both in the prevention of its eleva- dicina, Universidad de Buenos Aires, Argentina. tion after argon laser trabeculoplasty and in long-term control Supported by grants from Universidad de Buenos Aires, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), and of IOP in patients with glaucoma and ocular hyperten- 16–19 Fundacio´n Antorchas, Buenos Aires, Argentina. sion. A dual effect has been suggested as the mechanism Submitted for publication October 21, 1999; revised January 6, of the hypotensive action of brimonidine: a decrease in aque- 2000; accepted January 18, 2000. ous humor production and an increase in uveoscleral out- Commercial relationships policy: N. 19 Corresponding author: Ruth E. Rosenstein, Departamento de Bio- flow. Until now, no relationship has been established be- quı´mica Humana, Facultad de Medicina, UBA, Paraguay 2155, 5to P, tween this drug and trabecular GAGs. The purpose of the (1121), Buenos Aires, Argentina. [email protected] present study was to examine the presence of hyaluronidase

Investigative Ophthalmology & Visual Science, July 2000, Vol. 41, No. 8 2268 Copyright © Association for Research in Vision and Ophthalmology

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activity in the rabbit trabecular meshwork and its regulation by at 585 nm was measured keeping blanks for reagent and brimonidine. substrate. Heat-inactivated tissue extracts were used to assess the nonspecific release of N-acetylhexosamine. To determine pH activity profile, the pH of the reaction buffer was adjusted METHODS with acetic acid or NaOH. Hyaluronidase activity, expressed in milliunits, was defined as the amount of enzyme that causes the Reagents and Drugs release of 1 nanomole N-acetylglucosamine in 1 hour at 37°C. Hyaluronic acid, p-dimethyl-aminobenzaldehyde, 3-isobutyl- In our experimental conditions enzymatic degradation of hyal- methylxanthine (IBMX), 8-bromoadenosine 3Ј-5Ј-cyclic mono- uronic acid was linear with time up to 8 hours. phosphate (8-Br cAMP), and 2Ј-O-dibutyryladenosine 3Ј-5Ј-cy- clic monophosphate (dibutyryl cAMP) were obtained from Assay of cAMP Level Sigma (St. Louis, MO), and yohimbine was obtained from RBI Trabecular meshwork tissues were incubated for 30 minutes at (Natick, MA). Brimonidine tartrate was kindly supplied by 37°C in 3 ml HEPES-Tris buffer containing 0.5 mM IBMX, with Allergan-LOA (Buenos Aires, Argentina). or without 0.2% brimonidine, in the presence or absence of yohimbine (final concentration, 0.5 mM). The tissues were Animals and Tissues homogenized in 1 ml 0.5 mM IBMX and boiled for 2 minutes. Male albino rabbits (average weight, 2.5 Ϯ 0.3 kg) were anes- The homogenates were cooled and centrifuged at 5000g for 5 thetized with intravenous pentobarbital (40 mg/kg) and killed minutes at 4°C. The content of cAMP was measured in the by an air injection into the marginal vein of the ear. After death, supernatants by radioimmunoassay. Aliquots of samples or the eyes were quickly enucleated and placed in 0.25 M ice-cold standards were acetylated with acetic anhydride-triethylamine. sucrose containing 20 mM Tris-HCl buffer (pH 7.4). To isolate The acetylated products were mixed with [125I]-cAMP the trabecular meshwork tissues, the sclera was cut off radially (15,000–20,000 dpm, specific activity 140 mCi/millimole) and from the posterior pole to the equator to remove the vitreous, a rabbit antiserum kindly supplied by the National Institute of retina, choroid, and lens. After the tips of the ciliary process Diabetes and Digestive and Kidney Disease (1:5000 working had been excised, the iris was carefully cut from the ciliary solution) and incubated overnight at 4°C. The antigen–anti- body. An incision was made at the limbal level in the cornea body complexes were precipitated with ethanol at 4°C using and the cornea was cut off radially, leaving the scleral spur 2% bovine serum albumin as a carrier, centrifuged at 2000g for with the limbal sclera and including the corneoscleral and 20 minutes, and separated by aspirating supernatants. Radio- uveal portions of the trabecular meshwork. activity in the pellet was measured in a gamma counter. The range of the standard curve was 10–5000 femtomoles of cAMP. Hyaluronidase Assay System Protein content was determined by the method of Lowry et The trabecular tissues were incubated for 2 hours at 37°C in al.,22 using bovine serum albumin as the standard. HEPES-Tris in 3 ml buffer containing 140 mM NaCl, 5 mM KCl, All animal use procedures were in strict accordance with

2.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM the ARVO Statement for the Use of Animals in Ophthalmic and glucose, adjusted to pH 7.4 with Tris base, in the presence or Vision Research. absence of brimonidine tartrate, yohimbine, or cyclic adeno- sine monophosphate (cAMP) analogues (8-Br and dibutyryl cAMP). The final concentration of brimonidine was 0.2% wt/ RESULTS vol (4.3 mM). After the medium was removed, the trabecular Hyaluronidase activity was detected in the rabbit trabecular tissues were minced with scissors, homogenized in 3 ml 0.25 M meshwork, using the spectrophotometric assay described. Av- sucrose-Tris buffer (pH 7.4), and poured onto two layers of fine erage specific activity was 100 Ϯ 7 nanomoles/h ⅐ mg protein. gauze to filter off the tissue remnants. The filtrate was centri- The optimum pH for the trabecular hyaluronidase activity was fuged at 1000g for 10 minutes, and the pellet was discarded. determined by altering the buffer pH in the enzymatic assay. The supernatant was frozen and thawed five times and centri- When hyaluronidase activity was plotted as a function of pH, a fuged at 12,500g for 20 minutes. The resultant supernatant was major peak of activity was observed at pH 3.8, with a second dialyzed for 6 hours against 0.1 M acetate buffer (0.9% NaCl, minor peak in the neutral range (Fig. 1). pH 3.8). The nondialyzable material was concentrated in Cen- Brimonidine significantly increased hyaluronidase-specific tricon 10 concentrators (Amicon, Beverly, MA) and used as activity as shown in Figure 2. Yohimbine, although ineffective tissue extract (100–150 ␮g of protein/tube). The hyaluroni- itself, completely blocked the effect of brimonidine. Because dase assay was performed using the modified method of Aron- the decrease of cAMP levels is the mechanism classically asso- son and Davidson.20 The 0.5-ml reaction system contained 300 ciated with ␣ -adrenergic action, the effect of brimonidine on ␮g hyaluronic acid in 250 ␮l 0.1 M acetate buffer and tissue 2 this nucleotide level was assessed. Brimonidine significantly samples. After 3 to 5 hours at 37°C, the reaction was stopped decreased cAMP accumulation (Fig. 3). Yohimbine significantly by raising the reaction pH from 3.8 to 8.9 by adding 10 ␮l4N inhibited the effect of brimonidine on cAMP content. As NaOH and 100 ␮l 0.8 M potassium tetraborate solution (pH shown in Figure 2, 0.5 mM 8-Br or dibutyryl cAMP did not 9.2). The reaction mixture was assessed for the N-acetylhex- modify hyaluronidase activity. osamine end groups by the method of Reissig et al.,21 with N-acetylglucosamine (5–500 nanomoles) as the standard. After the pH was increased, the mixture was kept in a boiling water DISCUSSION bath for 3 minutes, cooled, and treated with 3 ml 1% p- dimethyl-aminobenzaldehyde reagent in glacial acetic acid con- Although the presence of hyaluronic acid in the trabecular taining 1.25% 10 N HCl for 50 minutes at 37°C. Optical density meshwork is well known, the mechanism of its clearance is not

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FIGURE 1. Rabbit trabecular meshwork hyaluronidase activity as a function of pH. Optimum activity is at pH 3.8 with a second minor peak at pH 7.

FIGURE 3. Tissues were incubated in the presence of IBMX with or without 0.2% brimonidine tartrate and yohimbine (0.5 mM). cAMP completely understood. In agreement with a former study by levels were assessed by radioimmunoassay. Brimonidine significantly 23 24 Mayer et al., Laurent and Reed reported that HA is not decreased nucleotide levels, with its effect reversed by yohimbine. significantly metabolized intracamerally but leaves the anterior Yohimbine itself had no effect. Data are mean Ϯ SEM (n ϭ 12 animals chamber gradually by the bulk flow. However, other investiga- per group). **P Ͻ 0.01, by Dunnett’s test. tors suggest that various tissues lining the anterior chamber digest the intracameral HA through an intralysosomal hyaluron- assay. Hayasaka and Sears, although using a method based on idase that hydrolyzes the N-acetylglucosamine bond. In fact, carbocyanine dye binding that has several limitations, reported hyaluronidase activity was measured in human vitreous25 and that lysosomal hyaluronidase activity in the inner layer of rabbit corneal endothelium26 by an enzyme-linked immunosorbent corneoscleral junction containing trabecular meshwork shows the highest specific activity among the corneoscleral tissues.27 Unlike that used in most studies of ocular hyaluronidase activ- ity, the methodology used in the current study allowed us to assess enzymatic activity in absolute units (e.g., the amount of enzyme that causes the release of 1 nanomole N-acetylglu- cosamine in 1 hour at 37°C) and therefore does not rely on exogenous hyaluronidase as a standard for quantification. This methodology has been successfully used for assessment of hyaluronidase activity in several systems, including rat liver,20 goat spermatozoa,28 and human trabecular cell cultures.29 The optimum hyaluronidase activity was in the acid range of pH 3.8 with a second minor activity peak at pH 7. Isolated hyaluron- idase from different sources also have an optimum pH in the acid range, indicative of a lysosomal origin. With respect to the eye, both cultured human trabecular meshwork cells,29 human cornea,26 and rabbit cornea and uvea27 have been shown to have acid hyaluronidase activity. The finding of endogenous hyaluronidase activity in rabbit trabecular meshwork further supports the hypothesis that this tissue can metabolize its own GAG products. Because the trabecular meshwork has been reported to have phagocytic activity,30,31 it seems likely that this enzyme may degrade the engulfed endogenous HA. The potential regulation of hyaluronidase by ocular drugs may pro- vide important clues to its function in physiological and patho- FIGURE 2. Effect of brimonidine on trabecular meshwork hyaluroni- logic conditions. dase activity. Tissues were incubated for 2 hours with or without 0.2% Brimonidine is emerging as a first-line therapy for primary brimonidine tartrate, yohimbine (0.5 mM), 8Br-cAMP (0.5mM), and open-angle glaucoma with a peak IOP-lowering efficacy com- dibutyryl cAMP (0.5 mM). Brimonidine significantly increased hyalu- ronidase-specific activity, whereas yohimbine, which is ineffective parable with that of timolol, but without an adverse cardiopul- itself, completely blocked the effect of brimonidine. Hyaluronidase- monary side effect, and it offers a more favorable systemic 32 specific activity was unchanged in the presence of both cAMP ana- safety profile than that of nonselective ␤-blockers. The logues. Data are mean Ϯ SEM (n ϭ 12 animals per group). **P Ͻ 0.01, present results indicate that 0.2% brimonidine significantly by Dunnett’s test. increases hyaluronidase-specific activity in the rabbit trabecu-

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lar meshwork. This concentration of brimonidine, although References high, is currently used to lower IOP in patients with ocular 1. Knepper PA, Farbman AI, Telser A. Aqueous outflow pathway hypertension and glaucoma.16 The effect of brimonidine was glycosaminoglycans. Exp Eye Res. 1981;32:265–277. completely blocked by yohimbine, an ␣2-selective antagonist 2. Acott TS, Westcott M, Passo MS, Van Buskirk EM. Trabecular that is ineffective itself. The intracellular events triggered by meshwork glycosaminoglycans in human and cynomolgus monkey brimonidine that could account for its effect on trabecular eye. Invest Ophthalmol Vis Sci. 1985;26:1320–1329. hyaluronidase activity remain to be established. Activation of 3. Segawa K. Ultrastructural changes of the trabecular meshwork in primary open glaucoma. Jpn J Ophthalmol. 1975;19:317–322. ␣2-adrenergic receptors inhibits adenylyl cyclase activity and decreases cAMP levels in a number of secretory and absorptive 4. Rohen JW. Why is intraocular pressure elevated in chronic simple glaucoma? Ophthalmology. 1983;90:758–769. epithelia, including ciliary epithelium of the eye, through a Gi 33 5. Fine BS, Yanoff M, Stone RA. A clinicopathologic study of four protein–dependent mechanism. The presence of functional cases of primary open-angle glaucoma compared to normal eyes. ␣2-adrenergic receptors has been demonstrated in cultured Am J Ophthalmol. 1981;91:88–105. human trabecular meshwork cells.34 Because brimonidine de- 6. Franc¸ois J, Benozzi J, Victoria–Troncoso V, Bohyn W. Ultrastruc- creased IBMX-induced cAMP accumulation in rabbit trabecular tural and morphometric study of corticosteroid glaucoma in rab- meshwork, with its effect blocked by yohimbine, it seems bits. Ophthalmic Res. 1984;16:168–178. likely that this type of adrenergic receptor is also present in 7. Barany EH, Scotchbrook S. Influence of testicular hyaluronidase on the resistance to flow through the angle of the anterior chamber. rabbit tissue. Acta Physiol Scand. 1954;30:240–248. It is known that stimulation of adenylate cyclase (e.g., by 8. Melton CE, DeVille WB. Perfusion studies on eyes of four species. ␤2-adrenergic agonists) leads to an increase in ocular outflow. Am J Ophthalmol. 1960;50:302–308. Therefore, it is expected that inhibition of cAMP synthesis by 9. Van Buskirk MS, Brett J. The canine eye: in vitro dissolution of the barriers to aqueous flow. Invest Ophthalmol Vis Sci. 1978;17:258– an ␣2-adrenergic agonist, as is the case with brimonidine, results in decreased outflow. However, it has been demon- 263. strated that brimonidine significantly decreases uveoscleral re- 10. Grant WM. Experimental aqueous perfusion in enucleated human 35 19 eyes. Arch Ophthalmol. 1963;69:783–801. sistance both in rabbit and human, contributing to its 11. Knepper PA, Farbman AI, Telser AG. Exogenous hyaluronidase and ocular hypotensive effect. Results in the current study may degradation of hyaluronic acid in the rabbit. Invest Ophthalmol account for this apparent discrepancy. If a decrease in cAMP Vis Sci. 1984;25:286–293. levels explains the brimonidine-induced increase in hyaluron- 12. Hubbard WC, Johnson M, Gong H, et al. Intraocular pressure and idase activity, a reduction of enzymatic activity in the presence outflow facility are unchanged following acute and chronic intra- of nucleotide analogues could be expected. Thus, because cameral chondroitinase ABC and hyaluronidase in monkeys. Exp Eye Res. 1997;65:177–190. trabecular hyaluronidase activity was unaffected by cAMP an- 13. Lerner LE, Polansky JR, Howes EL, Stern R. Hyaluronan in the alogues, it is possible that brimonidine’s effect could involve a human trabecular meshwork. Invest Ophthalmol Vis Sci. 1997;38: non–cAMP-mediated response. In fact, it has been demon- 1222–1228.

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