Characterization of adenylate cyclase activity in bovine and human corneal epithelium. Ronald J. Walkenbach, Roy D. LeGrand, and Ronald E.

Transcription

1 Characterization of adenylate cyclase activity in bovine and human corneal epithelium Ronald J. Walkenbach, Roy D. LeGrand, and Ronald E. Barr The properties of adenylate cyclase from bovine and human corneal epithelium were investigated. Adrenergic drugs ivere the most effective stimulatory agents tested in bovine tissue, causing greater activation than did fluoride. Isoproterenol was the most potent agonist, followed by epinephrine and norepinephrine. Phenylephrine and dopa.nine also stimulated adenylate cyclase through ^-adrenergic receptors at relatively high concentrations. Enzyme stimulation by all the adrenergic drugs tested was completely inhibited by 1 (JLM propranolol or 0.1 fjim timolol. The GTP analogue, GppNp, produced considerable activation and caused an augmented response when combined with isoproterenol, but not with fluoride. Prostaglandins E u E 2, or F 2a produced a small but significant stimulation over control which was not sensitive to propranolol inhibition. Adenylate cyclase from human corneal epithelium exhibited qualitatively similar characteristics to those of the bovine enzyme. Fluoride was the most effective stimulatory agent, followed by isoproterenol, phenylephrine, and dopamine. Prostaglandins failed to stimulate adenylate cyclase activity in human corneal epithelial preparations. Key words: adenylate cyclase, cyclic AMP, cornea, epithelium, isoproterenol, prostaglandins, phenylephrine cyclic AMP has been found to be an extremely important mediator of a wide variety of biological functions in many different tissues. Its role in the regulation of both synthesis and breakdown of glycogen has been firmly established 1 and has been strongly implicated in the control of lipolysis. 2 More recently, cyclic AMP has been suggested in the mediation of immunologic reaction mechanisms, 3 ' 4 From the Eye Research Foundation of Missouri, Inc., and the Departments of Ophthalmology and Pharmacology, University of Missouri School of Medicine, Columbia. Supported in part by U.S. Public Health Service grants EY-02597, EY-01604, and the Missouri Lions Eye Tissue Bank. Submitted for publication Aug. 6, Reprint requests: Dr. Ronald J. Walkenbach, Eye Research Foundation of Mo., Inc., 404 Portland, Columbia, Mo inflammatory response mechanisms, 3, 5) 6 collagenase activity, 7 ' 8 cell growth and mitotic rates, 9 ' 10 and glycosaminoglycan synthesis by fibroblasts. 11 " 13 All these cellular processes play important roles in corneal physiology. In addition, several corneal cell functions have already been shown to be regulated by cyclic AMP. Zadunaisky and co-workers 14 " 17 have described a chloride transport system in rabbit and frog corneal epithelium which is regulated by cyclic AMP. Epinephrine, prostaglandins, dibutyryl cyclic AMP, and phosphodiesterase inhibitors stimulated the transfer of chloride from the aqueous to tear side of the epithelium. This function has been associated with an increase in transparency 18 and thinning 19 of the cornea. Other investigators have demonstrated the chloride transport to be mediated through /3-adrenergic receptors. 20 The number of these receptor sites /80/ $ Assoc. for Res. in Vis. and Ophthal., Inc.

2 Volume 19 Number 9 Adenylate cyclase activity in corneal epithelium log [drug](m) Fig. 1. Stimulation of bovine corneal epithelial adenylate cyclase by various drugs. Activity was measured with varying concentrations of isoproterenol ( ), epinephrine (o), norepinephrine (A), GppNp ( ), phenylephrine ( ), dopamine (A), and NaF (o). has been shown to be affected by topical treatment of rabbit corneas with a /3- adrenergic agonist or antagonist. 21 Cyclic AMP also appears to be involved in the regulation of corneal epithelial cell division. 22 " 24 Drugs such as isoproterenol caused a transient inhibition of cell division in vivo and altered the circadian rhythm of mitosis. Despite the accumulation of information implicating cyclic nucleotides as an important mediator in corneal physiology, little attention has been given to the characterization of the key enzymes involved in cyclic AMP metabolism. In this paper we describe the properties of adenylate cyclase, the membranebound enzyme which synthesizes cyclic AMP from ATP, in bovine and human corneal epithelium. Methods and materials Bovine eyes were enucleated within 45 min after death at a local abattoir. Eyes were transported in ice-cold 0.9% NaCl and processed immediately. All preparative steps were performed in the cold. Corneal epithelial tissue was scraped from the intact eye and immersed in a solution of 0.25M sucrose, 10 mm tris (hydroxymethyl) aminomethane (Tris), and 10 mm EDTA, ph 7.5. This mixture was homogenized with six to eight passes in a Teflon-glass tissue grinder. Connective tissue was removed byfiltrationthrough a layer of surgical gauze before centrifugation at 100,000 X g for 60 min. The supernatant, containing cytoplasmic proteins and other soluble materials, was decanted. The pellet was resuspended in fresh buffer to a protein concentration of 5 to 10 mg/ml. This suspension was divided into small aliquots and stored at 70. Approximately 3 mg of epithelial particulate protein was recovered from each bovine cornea. Human eye tissue (donor ages 65 to 82) was obtained through the Lions Eye Tissue Bank, Columbia, Mo. Corneal epithelium was scraped from each intact eye within 16 hr after death, immersed in 250 /x\ of the above buffer, and stored at - 70 until analyzed. On the day of assay, tissue from 20 to 25 corneas was thawed, pooled, and homogenized, and the particulate fraction isolated as described for the bovine tissue. Each human cornea yielded approximately 0.3 mg of epithelial particulate protein. Adenylate cyclase activity was measured by use of the method of Salomon et al. 25 Final assay concentrations were 40 mm iv-2-hydroxyethyl-piperazine- W-2-ethanesulfonic acid (HEPES), 1.2 mm a- 32 P-ATP (5 to 10 cpm/pmol), 10 mm MgCl 2, 0.5 mm 3-isobutyl-l-methylxanthine, 2 mm cyclic AMP, 0.25 mg/ml bovine serum albumin, 25 mm creatine phosphate, and 0.01 U of creatine phosphokinase. Unless otherwise indicated, 75 to 100

3 1082 Walkenbach, LeGrand, and Barr Invest. Ophthalmol. Vis. Sci. September prostaglandin (/»M) Fig. 2. Activation of bovine corneal epithelial adenylate cyclase by prostaglandin s. Activity was measured with varying concentrations of prostaglandins E, ( ), E 2 (o), and F 2a (A). Values represent mean ± S.E. of six determinations. ixg of protein of enzyme preparation was assayed for 40 min at 30, ph 7.5, in a 75 (x\ volume. Cyclic a2 P-AMP was isolated by sequential chromatography on Dowex 50 and neutral alumina columns before measurement of radioactivity by standard liquid scintillation techniques. Protein was measured by the method of Lowry et al. 2G Radiolabeled ATP was purchased from ICN Pharmaceuticals, Irvine, Calif. Timolol used was Timoptic sterile ophthalmic solution, Merck, Sharp & Dohme, West Point, Pa. Other drugs and biochemicals were purchased from Sigma Chemical Co., St. Louis, Mo. Chemicals used were at least reagent grade. Results Bovine corneal epithelial adenylate cyclase. Preliminary experiments showed that bovine corneal epithelial adenylate cyclase exhibited kinetic properties similar to those of the enzyme from other tissues; that is, enzyme activity was proportional to protein concentration between 25 to 100 ixg per assay and to assay time for at least 40 min under basal conditions and when stimulated by NaF or isoproterenol. Activity was proportional to substrate concentrations up to 1 mm ATP, with half-maximal activity (K m ) exhibited at 0.2 mm under basal or stimulated conditions. Enzyme activation occurred through an increase in the number of active enzymes sites (V ma x)> with no change in substrate affinity. Maximal enzyme activity required the presence of 5 to 10 mm Mg Cl 2. Manganese ion was partially effective, but calcium ion inhibited activity at all levels tested (data not presented). The pharmacological regulation of adenylate cyclase in bovine corneal epithelium is summarized in Fig. 1. This graph shows the potency and efficacy of various drugs on enzyme activation. Isoproterenol, a /3-adrenergic agonist, was the most potent stimulatory agent tested. Epinephrine and norepinephrine, which exhibit both a-and /3- adrenergic agonist properties, also stimulated adenylate cyclase at relatively low concentrations. Phenylephrine and dopamine activated adenylate cyclase at higher drug levels. The nonmetabolizable analogue of guanosine triphosphate, 5' guanylylimidodiphosphate (GppNp), produced considerable enzyme stimulation between 0.05 and 10 fxm. Sodium fluoride stimulated activity between 1 and 5 mm but did not produce as great a maximal response as did the /3-adrenergic agonists. The following drugs failed to affect adenylate cyclase activity: carbachol (0.1 to 100 fxm), parathormone (0.1 to 10 fxm), vasopressin (0.1 to 10 /xm), histamine (0.1 to 100 mm), or adenosine (0.1 to 100 /xm). The effect of prostaglandins E x, E 2, and F 2a on adenylate cyclase activity is shown in Fig. 2. Each prostaglandin tested significantly increased activity over basal levels in a dosedependent manner, but the magnitude of stimulation was far less than that of the adrenergic drugs, GppNp, or fluoride. Stimulation of /3-adrenergic receptor-mediated adenylate cyclase activity can be specifically inhibited by antagonists such as propranolol. Fig. 3 shows the effect of propranolol and timolol (Timoptic) on isoproterenol-stimulated adenylate cyclase. Both drugs decreased activity to near basal levels. Propranolol exhibited half-maximal inhibition at 50 nm. Timolol was approximately 10-fold more potent than propranolol.

4 Volume 19 Number 9 Adenylate cyclase activity in corneal epithelium _ \ ^l I ^ \ X> \ \ \ s \ \ N \ \ 1 1 I I I I I >^_ N V x x\x \ s x xv I /i i J 1 -log [antagonist] (M) Fig. 3. Effect of/3-adrenergic antagonists on isoproterenol-stimulated bovine corneal epithelial adenylate cyclase. All assays contained 0.2 /xm isoproterenol and vaiying concentrations of propranolol ( ) or timolol (o). Data are presented as a percent of the activity expressed in the absence of antagonist. Table I. Effect of propranolol on bovine corneal epithelial adenylate cyclase stimulation by various agents Agents added None (basal) NaF, 5 mm GppNp, 10 jum PGE 2> 5 fim Isoproterenol, 0.2 fim Epinephrine, 1 /u,m Norepinephrine, 10 fim Phenylephrine, 100 /xm Dopamine, 100 /u.m NaF + isoproterenol NaF + PGE 2 NaF + GppNp GppNp + PGE 2 Isoproterenol + GppNp Isoproterenol + PGE 2 Isoproterenol + phenylephrine Enzyme activity Propranolol 4.7 : t dt db dt db dt db dt dt dt : t dt db db db ± 1.1 (pmol/mghnin)* +1 fxm propranolol 4.2 : t : t : b : t : b : b : b : b : b : b : b : b db : b : b : b 1.2 PGE 2 = prostaglandin E 2. * Values are mean ± S.E. of triplicate assays on four enzyme preparations. The mechanisms of bovine corneal epithelial adenylate cyclase activation were investigated by observing the effect of propranolol on the level of enzyme activation produced by the various stimulatory agents seen in Fig. 1. Table I shows that propranolol (1 //-M) had little or no effect on basal enzyme activity or on activity stimulated by F~, GppNp, or prostaglandin E 2. In contrast, propranolol caused essentially complete inhibition of adenylate cyclase activation by isoproterenol, epinephrine, norepinephrine, phenylephrine, or dopamine. No additional stimulation was observed when NaF was combined with isoproterenol, prostaglandin E 2, or GppNp. Combinations of GppNp and prostaglandin E 2 showed no augmented response over that with GppNp alone, but GppNp and isoproterenol produced further enzyme activation. The com-

5 1084 Walkenbach, LeGrand, and Barr Invest. Ophthalmol. Vis. Sci. September 1980 Table II. Human corneal epithelial adenylate cyclase Agents added None (basal) NaF, 5 mm GppNp, 10 ju-m Isoproterenol, 0.2 /AM Epinephrine, 1 /xm Epinephrine + propranolol (1 fim) Norepinephrine, 10 JU,M Phenylephrine, 100 (im Phenylephrine + propranolol Dopamine, 100 pim PGE,, 10 MM PGE 2, 10 nm PGF 2a> 10 /xm PGE = prostaglandin E; PGF = prostaglandin F. *Values are mean ± S.E. for six determinations. Enzyme activity (pmol/mg/min)* 2.6 ± : t : t : b dt dt dt dt dt ± ± ± ± 0.1 bination of prostaglandin E 2 and isoproterenol produced additional enzyme stimulation over isoproterenol alone, but combinations of other adrenergic drugs with isoproterenol caused no further response. Human cornea epithelial adenylate cyclase. Drug response characteristics of human cornea epithelial adenylate cyclase were examined with optimal assay conditions observed for the enzyme from bovine tissue. Table II shows that basal activity was lower than that of bovine enzyme, but fluoridestimulated activity was higher. Adrenergic drugs produced considerable activation, but it was far less than fluoride-stimulated activity. Enzyme stimulation by epinephrine and phenylephrine was sensitive to propranolol inhibition. The prostaglandins did not stimulate activity in human preparations. Histamine (^0.1 mm) had no measurable effect on basal or isoproterenol-stimulated activity (data not presented). Discussion An understanding of cyclic nucleotide metabolism in the cornea is of particular interest not only because it regulates corneal processes physiologically but also because the wide variety of topically administered drugs to the eye makes the cornea a clinically relevant model for studying pharmacologically induced perturbations of cyclic nucleotide metabolism. Particulate fractions of both bovine and human corneal epithelium exhibit relatively high adenylate cyclase activity. For example, isoproterenol-stimulated activity from either tissue is approximately twice that of antidiuretic hormone-sensitive adenylate cyclase from similarly prepared porcine kidney. 28 Adenylate cyclase from the two corneal sources exhibited properties generally similar to enzymes from other tissues in terms of their kinetic properties, substrateaffinity characteristics, divalent cation dependency, and fluoride activation. Maximal /3-adrenergic stimulation of the bovine enzyme resulted in activity greater than that produced by fluoride. This finding is unusual, but has been observed previously in several other tissues. 29 ' 30 Adenylate cyclase in both bovine and human corneal epithelium appears to be regulated predominantly by /3-adrenergic receptors. As expected, isoproterenol and epinephrine were the most potent agonists. Interestingly, both phenylephrine and dopamine were effective at relatively high concentrations (Fig. 1, Table I). Although these drugs are not considered /3-adrenergic agonists, each have been shown to activate /3-adrenergic-mediated adenylate cyclase at similar concentrations in other systems. 31 ' 32 Stimulation of corneal epithelial adenylate cyclase by phenylephrine may be clinically significant, in view of the widespread use of 10% (0.49M) phenylephrine (Neo-Synephrine) to produce maximal mydriasis. This concentration is 5000 times greater than the level needed to substantially activate human corneal epithelial adenylate cyclase in vitro (Table II), suggesting that clinical applications of topical phenylephrine could significantly elevate cyclic AMP in the cornea. It also seems plausible that /3- adrenergic influences may play a role in the untoward effects of high doses of topical phenylephrine on corneal epithelium. 33 ' 34 Bovine corneal epithelial adenylate cyclase showed small but significant increases in activity with all of the prostaglandins tested. Typically, activity was increased 60% to 70% over control (Fig. 2). Although this level of stimulation may seem small in comparison to the fivefold to sixfold increase produced by

6 Volume 19 Number 9 Adenylate cyclase activity in corneal epithelium 1085 /3-adrenergic agonists, it nevertheless could be physiologically significant. Corneal epithelial chloride transport was stimulated by prostaglandins Ei, E 2, or F 2a in frog corneal epithelium, 18 but this species may possess greater prostaglandin-sensitive adenylate cyclase than the bovine tissue. It remains unclear whether the lack of prostaglandin response in human corneal epithelial adenylate cyclase represents a species difference or a lack of prostaglandin receptor stability during the time between enucleation and enzyme preparation. The former seems more probable since bovine corneas do not lose any prostaglandin-sensitive adenylate cyclase if eyes are stored at 4 for 16 hr in a moist chamber before enzyme preparation (data not presented). These data indicate that cyclic AMP synthesis in bovine and human corneal epithelium is controlled primarily by /3-adrenergicmediated adenylate cyclase, which is consistent with previous reports of increased cyclic AMP content in rabbit corneas by epinephrine. 15j 16> 21 Bovine tissue also contains separate prostaglandin receptors. Activation of adenylate cyclase appears to be the initial step in the stimulation of chloride transport, 14 " 19 inhibition of collagenase, 8 and alterations in mitotic activity 22 " 24 in corneal tissue. REFERENCES 1. Cohen P: The role of cyclic AMP-dependent protein kinase in the regulation of glycogen metabolism in mammalian skeletal muscle. Curr Top Cell Regul 14:117, Huttenen J, Steinberg D, and Mayer S: Protein kinase activation and phosphorylation of a purified hormone-sensitive lipase. Biochem Biophys Res Commun 41:1350, Bourn HR, Lichtenstein LM, Melmon KL, Henney CS, Weinstein Y, and Shearer GM: Modulation of inflammation and immunity by cyclic AMP. Science 184:19, Parker CW, Sullivan TJ, and Wedner JH: Cyclic AMP and the immune response. Adv Cyclic Nucleotide Res 4:1, Weissman G, Zurier P, and Zurier RB: Effect of cyclic AMP on release of lysosomal enzymes from phagocytes. Nature 231:131, Lichenstein LM: The role of cyclic AMP in inflammation. In Cyclic AMP, Cell Growth and the Immune Response, Braun W, Lichtenstein LM, and Parker C, editors. New York, 1974, Springer- Verlag, pp Koob TJ and Jeffrey JJ: Hormonal regulation of collagen degradion in the uterus: inhibition of collagenase expression by progesterone and cyclic AMP. Biochim Biophys Acta 354:61, Berman MB, Cavanaugh HD, and Gage J: Regulation of collagenase activity in ulcerating cornea by cyclic AMP. Exp Eye Res 22:209, Burger MM, Bombik BM, Breckenridge B, and Sheppard JR: Growth control and cyclic alterations of cyclic AMP in the cell cycle. Nature (New Biol) 239:161, Pastan IH, Johnson GS, and Anderson WB: Role of cyclic nucleotides in growth control. Annu Rev Biochem 44:491, Goggins JF, Johnson GS, and Pastan I: The effect of dibutyryl cyclic adenosine monophosphate on synthesis of sulfated acid mucopolysaccharides by transformed fibroblasts. J Biol Chem 247:5759, Schonhofer PS, Peters HD, Karzel K, Dinnendahl V, and Westhofen P: Influence of antiphlogistic drugs on prostaglandin Ei stimulated cyclic 3', 5'-AMP levels and glycosaminoglycan synthesis in fibroblast tissue cultures. Pol J Pharmacol Pharm 26:51, Peters H, Karzel K, Padberg D, Schonhofer P, and Dinnendahl D: Influence of prostaglandin Ei on cyclic 3', 5'-AMP levels and glycosaminoglycan secretion of fibroblasts cultured in vitro. Pol J Pharmacol Pharm 26:41, Chalfie M, Neufield AH, and Zadunaisky JA: Action of epinephrine and other cyclic AMP-mediated agents on the chloride transport of the frog cornea. INVEST OPHTHALMOL 11:644, Klyce SD, Neufeld AH, and Zadunaisky JA: The activation of chloride transport by epinephrine and db-cyclic AMP in the cornea of the rabbit. INVEST OPHTHALMOL 12:127, Zadunaisky JA, Lande MA, Chalfie M, and Neufeld AH: Ion pumps in the cornea and their stimulation by epinephrine and cyclic AMP. Exp Eye Res 15:577, Buck MG and Zadunaisky JA: Stimulation of ion transport by ascorbic acid through inhibition of 3', 5'-cyclic AMP phosphodiesterase in the corneal epithelium and other tissues. Biochim Biophys Acta 389:251, Beitch BR, Beitch I, and Zadunaisky JA: The stimulation of chloride transport by prostaglandins and their interaction with epinephrine, theophylline and cyclic AMP in the corneal epithelium. J Membr Biol 19:381, Klyce SD: Transport of Na, Cl, and water by the rabbit corneal epithelium at resting potential. Am J Physiol 228:1446, Montoreano R, Candia OA, and Cook P: a and /3-adrenergic receptors in the regulation of ionic transport in frog cornea. J Physiol 230:1487, Neufeld AH, Zawitowski KA, Page ED, and Bromberg BB: Influences on the density of/3-adrenergic

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