Alpha ^Adrenoceptors in Human Corneal Epithelium

Investigative Ophthalmology & Visual Science, Vol. 32, No. 12, November 1991 Copyright © Association for Research in Vision and Ophthalmology Alpha ^Adrenoceptors in Human Corneal Epithelium Ronald J. Walkenbach,*t Guo-Sui Ye,* Peter 5. Reinach,^: and Frances Boney* Specific binding of the potent, selective alphaj-adrenoceptor antagonist 3H-prazosin was demonstrated in cultured human corneal epithelial cells. Specific binding of the radioligand was concentration-dependent between 0.5 and 6 nM, with apparent saturation of receptor sites seen at higher concentrations. The cells exhibited a maximum binding capacity for 3H-prazosin of 225 fmol/mg of cellular protein and a dissociation constant of 2 nM. The binding of 3H-prazosin was competitive with known alpha,-adrenoceptor ligands and was reversible. Epithelium of intact human corneas also exhibited specific 3H-prazosin binding, as did cultures of bovine and rabbit corneal epithelium. The alpha-adrenergic agonist methoxamine significantly stimulated phosphatidylinositol 4,5-bis- phosphate hydrolysis, measured as myoinositol trisphosphate accumulation in cultures of human corneal epithelium. This stimulation was inhibited by the presence of prazosin during the assays. These findings indicate the existence of specific, reversible, high-affinity receptors for alpha,-adrenoceptors that regulate inositol phosphate turnover in human, rabbit, and bovine corneal epithelial cells. Invest Ophthalmol Vis Sci 32:3067-3072,1991 The cornea is innervated by adrenergic nerve late inositol phosphate turnover in human corneal epi- fibers,1"3 but their role(s) in corneal physiology re- thelial cells, a finding analogous to that previously main poorly understood. The existence of beta-adren- reported in rabbit corneal epithelium. oceptors on corneal epithelial cells has been estab- 4 5 Methods and Materials lished ' and shown to be predominately of the beta2 subtype.6 Corneal epithelial beta-adrenoceptors have Rabbit and bovine eyes were obtained from local been associated with stimulation of adenylate cyclase slaughterhouses within 2 hr after the animals were 7 9 and cyclic AMP-dependent protein kinase, " chlo- killed. The eyes were kept on ice for up to 4 hr more, 10 14 ride secretion, " as well as inhibition of mitotic until further processing occurred. Human eyes were 15 8 rates and glycogen synthase activity. obtained from the Missouri Lions Eye Tissue Bank. The existence of alpha-adrenoceptors on corneal ep- The corneas of human eyes were dissected within 12 ithelial cells is less well understood. Some preliminary hr postmortem and stored in Dexsol (Chiron Ophthal- reports using broken cell tissue preparations have sug- mics, Irvine, CA) or a similar medium composed of gested the absence of alpha-adrenoceptors,1617 M-199 tissue culture medium supplemented with whereas other studies have used drugs with alpha- 1.35% chondroitin sulfate, 1% dextran (40,000 kDa), adrenoceptor agonist properties to demonstrate stimu- 17 raM Na bicarbonate, 20 mM HEPES buffer, 12 lation of ion transport in frog and inositol phosphate brought to a final pH of 7.4 with 1 N NaOH. Corneas 18 turnover in rabbit corneal epithelium. were stored at 4°C for up to 72 hr in one of these The direct radioligand binding studies shown here media before initiation of tissue fractionation, cell indicate that intact corneal epithelial cells from rab- culture, or binding experiments. bit, bovine, and human tissue exhibit high-affinity, Particulate fractions of native corneal epithelium specific alpha radrenoceptors. These receptors regu- from rabbit, bovine, or human corneas were prepared as previously described.19 Briefly, the corneas were rinsed with an ice-cold solution containing 10 mM From the *Missouri Lions Eye Research Foundation, Columbia, K2HPO4 in 0.9% NaCl (PBS), and the corneal epithe- Missouri; the f Departments of Ophthalmology and Pharmacology, University of Missouri, Columbia, Missouri; and the tDepartment lium was removed from the isolated cornea (human) of Physiology and Endocrinology, Medical College of Georgia, Au- or eye (bovine and rabbit) with a scalpel blade. Tissue gusta, Georgia. was homogenized in 1 ml per cornea of 25 mM gly- Supported by National Institutes of Health grants EY 02597 and cylglycine buffer (pH = 7.6) with a Teflon/glass tissue EY 04795. grinder in the cold. The homogenate was centrifuged Submitted for publication: March 20, 1991; accepted June 17, 1991. at 50,000 X g for 30 min at 4°C. The pellet was resus- Reprint requests: Ronald J. Walkenbach, PhD, The Missouri pended in fresh buffer, and the centrifugation and re- Lions Eye Research Foundation, 404 Portland Street, Columbia, suspension steps were repeated to produce each epithe- MO 65201. lial particulate fraction. 3067 Downloaded from iovs.arvojournals.org on 09/28/2021 3068 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1991 Vol. 32 Rabbit, bovine, or human coraeal epithelial cells as the difference between the measured total 3H-pra- were cultured in six-well multiplates as described pre- zosin bound and nonspecific bound in each set of par- viously.19 Briefly, epithelial tissue pieces (with some allel assays. residual stroma) were placed in 5 ml per well as Ea- Binding studies with intact human corneas were gle's minimum essential medium (MEM) with D-va- performed analogously except that incubations were line to inhibit keratocyte contamination of the epithe- performed in culture media. After incubation, the lial cultures20 and 10% newborn calf serum. Media corneas were rinsed briefly with ice-cold PBS, and the were changed after 1 week of culture and twice weekly epithelium was removed quickly with a surgical scal- thereafter. Each well typically contained 150-200 ng pel blade and placed in 2 ml of 2 N NaOH. After of cellular protein when used for experiments after digestion, the samples were neutralized before mea- 3-4 weeks of culture. suring their protein and radioactivity. Paniculate fractions of cultured epithelium were Binding protocols using intact cultured cells were prepared as described except that a rubber spatula was identical except that 1 ml of 1 N NaOH was added to used to remove cells from the wells. each well after the final PBS rinse. The potent alphaj-adrenoceptor antagonist 3H- Inositol phosphate turnover experiments in cul- prazosin (87 Ci/mmol; NEN Research Products, Bos- tured human corneal epithelial cells were performed ton, MA) was used to assess receptor binding activity using the basic technique described by Martin.21 The in these tissue preparations. The protocol used for growth medium was removed and the cells washed binding to epithelial particulate fractions was analo- several times with inositol-free minimum essential gous to that described for 3H-quinuclidinyl benzilate medium (IFMEM). This medium was prepared by binding to muscarinic cholinoceptors in this tissue.19 mixing Hank's salt solution (Sigma Chemical Co., St. Briefly, total binding of the radioligand was assessed Louis, MO) with MEM amino acid mixture (Sigma) by incubating the particulate fractions with the indi- and adding the individual vitamins as listed by the cated concentration of 3H-prazosin in 25 mM glycylg- MEM formulation. The cells were cultured for 48 hr lycine buffer, pH = 7.6 at 37°C for 30 min (unless with 3 ml of fresh IFMEM, supplemented with 2% otherwise indicated). The assays were filtered and dialyzed sterile calf serum (Sigma) and 33 nM 3H- washed free of unbound radioligand with three 5-ml myoinositol (3H-inositol; 15.6 Ci/mmol, NEN Re- aliquots of ice-cold buffer. Each filter was placed in 10 search Products). The labeling medium was removed, ml of Scintiverse BOA cocktail (Fisher Scientific, and the cells were washed with PBS and preincubated Springfield, NJ), shaken for at least 1 hr in the dark, for 5 min at 37°C with serum-free IFMEM with 10 and counted by standard liquid scintillation tech- mM LiCl to block dephosphorylation of inositol-1- 3 22 niques. Nonspecific binding of H-prazosin was mea- PO4 to inositol. After the preincubation period, the sured by running parallel assays with 100 nM norepi- medium was replaced with fresh serum-free IFMEM nephrine added to the reaction mixtures during the containing the drugs indicated in Table 1 and incu- incubation. Specific binding of 3H-prazosih is defined bated for an additional 5 min. Reactions were termi- Table 1. Comparison of 3H-prazosin binding in different tissue preparations of corneal epithelium from the rabbit, bovine, and human 3H-prazosin bound (fmol/mg) Tissue preparation Total Nonspecific Specific Significance Rabbit Fresh, particulate fraction 435 ± 34 456 ± 33 <0 NS Cultured, particulate fraction 189 ±23 193 ± 15 <0 NS Cultured, intact cells 295 ± 15 259 ± 19 36 P < 0.05 Bovine Fresh, particulate fraction 243 ±31 238 ± 25 5 NS Cultured, particulate fraction 210 ± 17 201 ± 27 9 NS Cultured, intact cells 299 ± 25 154 ±20 145 P < 0.005 Human Fresh, particulate fraction 117± 13 126 ± 19 <0 NS Cultured, particulate fraction 165 ±21 155 ± 18 10 NS Cultured, intact cells 275 ± 36 182 ±33 93 P<0.01 Fresh, intact cornea 105 ± 7 64 ± 13 41 P < 0.01 Intact cells or particulate fractions were assayed using 2 nM 3H-prazosin 20 assays, using tissue from at least three different harvest dates. Statistical without or with 100 jtM norepinephrine. Each tissue preparation's total and significance was determined using the Student's t-test for unpaired samples. nonspecific 3H-prazosin binding value represents the mean ± SEM of 12 to Protein levels ranged from 75 to 100 ng per assay. Downloaded from iovs.arvojournals.org on 09/28/2021 No. 12 ALPHA,-ADRENOCEPTOR5 IN HUMAN CORNEAL EPITHELIUM / Wolkenboch er ol 3069 nated by aspirating the incubation medium and add- 200 ing 1 ml of ice-cold 10% HC1O4 to each well. The cells in the wells were frozen, thawed, and kept on ice for 30 min to complete the extraction of 3H-inositol phos- phates from the cells, then decanted.

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