120 Reports. Adenylate cyclase activity in bovine and human corneal endothelium. RONALD J.

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1 120 Reports Invest. Ophthalmol. Vis. Sci. January 1982 mors vs. control eyes) was obtained at prolonged intervals after administration. This coincides with the theory that the preferential localization of HpD in malignant tissue is related to the enhanced retention of the drug in tumor tissue compared with surrounding normal tissue. 3 It is unclear as to why HpD is preferentially retained in tumor tissue, but it has been suggested that the high vascular permeability together with the lack of adequate lymphatic drainage of tumors may contribute to this localization. 9 Although several of the characteristics of the human retinoblastoma are retained when grown in the nude mouse, it is necessary to emphasize that this tumor model differs from the original both in its environment (anterior chamber) and presumably in its growth rate. Therefore the uptake kinetics of HpD in the clinical case of retinoblastoma may differ from that observed in the nude mouse model. Current treatment modalities for intraocular tumors offer only limited success and in many patients enucleation is necessary. A treatment such as HpD PRT (which has been reported to be beneficial either as a primary treatment modality or as a follow-up treatment in conjunction with radiation therapy) may prove to be useful in selectively destroying intraocular tumors. In addition to the localized uptake of HpD in ocular tumors, it has been reported that HpD PRT can produce marked tumor toxicity in the nude mouse-human retinoblastoma model 10 and can be lethal to retinoblastoma cells (Y-79 and WERI-Rbl) in vitro. 11 Preclinical evaluation (ocular tissue toxicity, lightdelivery development, and tumor treatment) of ocular HpD PRT is currently being pursued with the pigmented rabbit and the Greene melanoma tumor model. It is hoped that these studies will lead to the safe and effective use of HpD PRT in the clinical treatment of intraocular tumors. We thank Ms. Carla Rother for assistance in the preparation of this manuscript. From the Division of Ophthalmology (C. J. G., A. L. M.) and Division of Hematology Oncology (C. J. G., N. R., C. M., W. F. B.), Childrens Hospital of Los Angeles, Clayton Center for Ocular Oncology, Los Angeles, Calif. This work was performed in conjunction with the Clayton Foundation for Research. Submitted for publication April 28, Reprint requests: Dr. Charles J. Gomer, Clayton Center for Ocular Oncology, Childrens Hospital of Los Angeles, 4650 Sunset Blvd., Los Angeles, Calif Key words: hematoporphyrin derivative, retinoblastoma, nude mice, photoradiation therapy, hematoporphyrin tissue uptake REFERENCES 1. Dougherty TJ, Kaufman JE, Goldfarb A, Weishaupt KR, Boyle DG, and Mittleman A: Photoradiation therapy for the treatment of malignant tumors. Cancer Res 38:2628, Dougherty TJ, Lawrence G, Kaufman JE, Boyle DG, Weishaupt KR, and Goldfarb A: Photoradiation in the treatment of recurrent breast carcinoma. JNCI 62:231, Dougherty TJ, Weishaupt KR, and Boyle DG: Photoradiation therapy of malignant tumors. In Principles and Practices of Oncology. DeVita V, Helman S, and Rosenberg S., editors. Philadelphia, J. B. Lippincott Co. (in press). 4. Gomer CJ and Dougherty TJ: Determination of 3 H and 14 C hematoporphyrin derivative distribution in malignant and normal tissue. Cancer Res 39:146, Weishaupt KR, Gomer CJ, and Dougherty TJ: Identification of singlet oxygen as the cytotoxic agent in photodestruction of a murine tumor. Cancer Res 36:2326, Cunningham RD and Henderson JW: Experimental evaluation of hematoporphyrin in the detection and management of intraocular tumors. Am J Ophthalmol 61:36, Krohn DL, Jacobs R, and Morris DA: Diagnosis of model choroidal malignant melanoma by hematoporphyrin derivative fluorescence in rabbits. INVEST OPHTHALMOL 13:244, Benedict WF, Dawson JA, Banerjee A, and Murphree AL: The nude mouse model for human retinoblastoma: a system for evaluation of retinoblastoma therapy. Med Pediatr Oncol 8:391, Bugelski PJ, Porter CW, and Dougherty TJ: Autoradiographic distribution of hematoporphyrin derivative in normal and tumor tissue of the mouse. Cancer Res (in press). 10. Benedict WF, Lingua RW, Doiron DR, Dawson JA, and Murphree AL: Tumor regression of human retinoblastoma in the nude mouse following photoradiation therapy: a preliminary report. Med Pediatr Oncol 8:397, Sery TW: Photodynamic killing of retinoblastoma cells with hematoporphyrin and light. Cancer Res 39:96, Adenylate cyclase activity in bovine and human corneal endothelium. RONALD J. WALKENBACH AND ROY D. LEGRAND. Adenylate cyclase activity was measured in particulate fractions prepared from bovine and human corneal en /82/ $00.50/ Assoc. for Res. in Vis. and Ophthal., Inc.

,U u0 0.4 0.8 1.2 1.6 2.0-12 -6 0 6 12 18 v u r ATP (mm) Fig. 1. A, Bovine corneal endothelial adenylate cyclase activity as a function of ATP concentration in the presence of no stimulato~y agents (e) (basal activity), 0.

2 ,U u v u r ATP (mm) Fig. 1. A, Bovine corneal endothelial adenylate cyclase activity as a function of ATP concentration in the presence of no stimulato~y agents (e) (basal activity), 0.5 pm isoproterenol (o), or 5.0 mm NaF (A). B, Double-reciprocal analysis of the relationship between enzyme velocity (V) and substrate concentration (S). Symbols used are the same as in A. clothelium. Bovine enzyme activity was stimulated by fluoride, 5'-guanylylimi~lodiphosphate (GppNp) and P adrenergic agonists. Drug stimulation patterns of the lairman enzyme appeared qualitatively siinilar, but activities were generally higher than those of the bovine enzrpne. This studtl demonstrates the existence of a rc:ceptor-~nediated adenylate cyclase system in bovine and human corneal endothelium and suggests a possible role for cyclic AMP in corneal endothelial physiology. (li\l~~~l' OI~ITIIAL~\IOL Table I. Bovine corneal endothelial adenylate cyclase activity* Assay conditions VIS VIS 22: , 2982.) Control (basal) NaF (5 mm) The role of cyclic AMP in the cornea has gen- GPPNP (1 PM) 5.6k erated much interest in recent vears. but attention Epinephrine (1 pm) ,, Adenylate cyclase activity (pmoles cyclic AMP fmned lmg protein linin) Without proprnnolol With 1 pm has focused mainly on the effects of the cyclic nu- l"proterenol (0-2 pm) 4.5 O a 7 Phenylephrine (100 pm) cleoticle on corneal epithelium. Neufeld and Dopamine (100 pm) tO.6 Sears' found no evidence of cyclic AMP synthesis Prostaglandin E, (1 p ~ ) in monkey cornea endothelial tissue. Dikstein2 reported that drugs which alter cyclic AMP levels in *Each value represents the mean t. S.E. of ten determinations, many tissues failed to affect the rate of fluid transcalculated from assays of three separate tissue preparations. port by rabbit corneal endothelium. More recently, however, our laboratory has observed both abattoir within 1 hr after death or from human adenylate cyclase" and cyclic AMP-dependent eyes obtained through the Missouri Lions Eye protein kinase activities4. in bovine and human Tissue Bank, Columbia, Mo.; within 18 hr after corneal endothelium. Whikehart and Fletcher6 enucleation. Eyes were maintained at 2' to 4' until have also reported the presence of cyclic AMP as used. Particulate fractions of bovine corneal enwell as cyclic GMP in fresh and cultured rabbit dothelium were prepared by removing all corneal corneal endothelial tissue. We have therefore epithelium from the intact eye, excising the corstudied the pharmacologic regulation of corneal nea, and scraping its endothelial surface with a endothelial adenylate cyclase to better understand spatula while submerged in 8 ml of ice-cold 0.25M the possible roles of cyclic AMP in this tissue and sucrose, 10 mm Tris, 1 mm EDTA (STE),. ph to assess the feasibility of pharmacologic manipu- Endothelial tissue was homogenized in a Teflonlation of. the cyclic AMP system in corneal en- glass tissue grinder and centrifuged at 100,000 x g dothelium for therapeutic benefit. for 30 min. The pellet was resuspended in 8 ml of Methods. Enzyme preparations were made fresh STE buffer and centrifuged again. This pellet from 60 to 90 bovine eyes obtained from a local was resuspended in 0.1 ml of STE per cornea and

3 122 Reports Invest. Ophthaltnol. Vis. Sci. Janua y [activator] (ym) --- Fig. 2. Stimulation of bovine corneal endothelial adenylate cyclase by various concentrations of isoproterenol (a), GppNp (o), phenylephrine (A) and dopamine (A). Data are expressed as percent of the activity observed when preparations were assayed with 5 mm NaF. stored at -20' in small aliquots until analyzed. Appqoximately 200 pg of particulate protein' were recovered. per bovine cornea. Human corneal endothelial fractions were prepared by the same.method, except that endothelial tiss'ue was scraped in 2 ml of STE buffer per pair of eyes, and this slurry was stored at,,-20" until tissue from 24 corneas had accumulated. These samples were then thawed, pooled, homogenized, fractionated as described above, and assayed immediately. Approximately 30 pg of particulate protein were recovered per human cornea. Adenylate cyclase activity was measured directly by recovering cyclic [32P]AMP synthesized from c~-[~~p]atp, ' with assay conditions and protein measurements as previously de~cribed.~ Assays were performed in the presence of 0.5 mm 3-isobutyl-1-methylxanthine to.inhibit cyclic nucleotide phosphodiesterase activity. Radiochemicals were purchased from ICN Pharmaceuticals, Irvine, Calif. GppNp was purchased from P-L Biochemicals, Milwaukee, Wisc. Other reagents were obtained from Sigma Chemical Co., St. Louis, Mo. Resttlts Bovine corneal endothelial adenylate cyclase. Preliminary experiments showed that the characteristics of corneal endothelial adenylate cyclase were very similar to those from other tissues. Activity was found only in particulate'fractions and was proportional to assay time and enzyme protein up to 50 min and 100 pg protein. Substrateactivity relationships for bovine corneal endothelid adenylate cyclase are shown in Fig. 1, A. Half-maximal enzyme activity occurred at 0.1 mm ATP, and maximal activity was expressed at approximately 1.2 m M. Double-reciprocal analysis of these data suggest that enzymatic stimulation by isoproterenol or fluoride ion occurred through an increase in the number of active enzyme sites, with no change in substrate affinity (Fig. 1, B). Magnesium ion (10 mm) was required for optimal enzyme expression. Manganese ion was partially effective, but calcium ion inhibited activity. The metal ion characteristics were valid whether the enzyme was at a basal level or activated by stirnulatory agents. The pharmacologic regulation of bovine corneal endothelial adenylate cyclase is described in Table I. Fluoride ion-stimulated activity was about fivefold over control. The nonmetabolizable analog of GTP, 5'-guanylylimidodiphosphate (GppNp); increased enzyme activity more than threefold above control. These two agents are generally thought to increase adenylate cyclase activity by mechanisms not involving receptors, and their stimulations

Volume 22 Nutnber 1 Reports 123 were not affected by the p-adrenergic antagonist,. Epinephrine and isoproterenol, both potent P-adrenergic agonists, stimulated activity almost threefold.

4 Volume 22 Nutnber 1 Reports 123 were not affected by the p-adrenergic antagonist,. Epinephrine and isoproterenol, both potent P-adrenergic agonists, stimulated activity almost threefold. Phenylephrine and dopainine are not considered P-adrenergic agonists, but each stimulated activity approximately twofold at relatively high concentrations. Propranolol (1 pm) completely blocked stimulations by all four adrenergic agents, indicating that P-adrenergic receptors were mediating the response of each drug. Prostaglandins El, E2, or F,,, parathormone, or vasopressin (510 pm) failed to alter enzyme activity. The potency and efficacy of various stimulatory agents on adenylate cyclase activity are shown in Fig. 2. Isoproterenol and GppNp were fairly potent activators, expressing maximal effects at less than 1 pm. In contrast to the previous agents, phenylephrine and dopamine required approximately 1 pm levels to initiate a response and greater than 100 pm to achieve a maximal effect. Human corneal endothelial ahnylate cyclase. Adenylate cyclase activity was measured in human corneal endothelial particulate fractions with identical drug treatments and other assay conditions as used for the bovine enzyme. Table I1 shows that the human enzyme activity was several times higher than its bovine counterpart, but the patterns of drug stimulation were qualitatively similar. Propranolol did not affkct basal, GppNp-, or fluoridestimulated activity but completely inhibited epinephrine stimulation. Discussion. Bovine and human corneal endothelium exhibit drug-stimulated adenylate cyclase activity, which appears to be regulated primarily by P-adrenergic receptors. Corneal endothelial adenylate cyclase differs from epithelial activity8 in several respects. In general, enzyme specific activities are higher in epithelial tissue. Adrenergic drugs stimulate epithelial adenylate cyclase to a greater degree than does fluoride, but these agents expressed only 30% to 40% of the maximal fluoride stimulation in endothelial tissue. Prostaglandins stimulate bovine epithelial adenylate cyclase approximately twofold but have no effect on the endothelial enzyme. These differences show that the activity measured in these experiments was not contaminant epithelial enzyme but demonstrate that corneal endothelial cells contain their own receptor-mediated enzyme system for synthesizing cyclic AMP. This suggests that the cyclic AMP response to an endogenous or exogenous agonist could be quite different in the corneal endothelium than in the epithelium. Many investigators have employed whole tissues in studies of cyclic AMP effects on the cornea. Because of the relative quantities of epithelial, Table 11. Human corneal endothelial adenylate cyclase activity* Assay conditions Adenylate cyclase activity (pmoles cyclic AMP formed lmg protein lmg) Without - - With 1 phi Control (basal) NaF (5 mm) GPPNP (1 PM) Epinephrine (1 pm) Phenylephrine (100 pm) Dopamine (100 pm) Prostaglandin E, (1 pm) *Each value represents the mean + S.E. of six determinations calculated from assays of two separate corneal endothelial preparations. keratocyte, and endothelial cells, whole cornea measurements of cyclic AMP probably reflect only epithelial responses. A lack of quantitative importance in whole corneal cyclic AMP measurements does not, however, indicate a lack of physiologic importance within keratocyte or endothelial cells. Intracellular concentrations of cyclic AMP could rise significantly in these corneal cells in response to synthesis of very small quantities ofcyclic AMP. Corneal endothelium exhibits adenylate cyclase, cyclic AMP-dependent protein kina~e,~ and phosphodiesterase activities (unpublished results). The presence of these enzymes suggests that cyclic AMP plays one or more roles in the regulation of corneal endothelial physiology. These roles are yet to be determined. Dikstein2 observed no direct effect of agents that should have increased corneal cyclic AMP on rabbit corneal endothelial fluid pump rates. Cyclic AMP may play a more subtle role in the corneal endothelium. For example, cyclic nucleotides have been shown to play a role in cell proliferation in many cell types,9 including corneal epithelium. lo It seems plausible that these agents may be involved in the corneal endothelium's curious inability to proliferate in vivo in several species and its ability to do so in vitro. If so, the cyclic nucleotide systems may provide a basis for pharmacologic treatment of corneal edema caused by low endothelial-cell density, a major cause of corneal blindness. From The Eye Research Foundation of Mo., Inc., and the Departments of Pharmacology and Ophthalmology, University of Missouri Health Sciences Center, Columbia, Mo. Supported in part by U.S.P.H.S. grant EY-02597, the Missouri Lions Eye Tissue Bank,

124 Reports Invest. Ophthalmol. Vis. Sci. January 1982 and Research to Prevent Blindness. Submitted for publication Nov. 3, 1980. Reprint requests: Ronald J. Walkenbach, Ph.D.

5 124 Reports Invest. Ophthalmol. Vis. Sci. January 1982 and Research to Prevent Blindness. Submitted for publication Nov. 3, Reprint requests: Ronald J. Walkenbach, Ph.D., Eye Research Foundation of Mo., Inc., 404 Portland, Columbia, Mo Key words: adenylate cyclase, cyclic AMP, cornea, corneal endothelium REFERENCES 1. Neufeld AH and Sears ML: Cyclic AMP in ocular tissues of monkey, rabbit and human. INVEST OPHTHALMOL 13:475, Dikstein S: Efficiency and survival of the corneal endothelial pump. Exp Eye Res 15:639, Walkenbach RJ and LeCrand RD: Adenylate cyclase activity in the bovine and human cornea. INVEST OPHTHALMOL VIS SCI 18(ARVO Suppl.):105, Walkenbach RJ and Barr RE: Cyclic AMP-dependent protein kinase in the bovine and human cornea. INVEST OPHTHALMOL VIS SCI 17(ARVO Suppl.):189, Walkenbach RJ, LeGrand RD, and Barr RE: Distribution of cyclic AMP-dependent protein kinase in the bovine cornea. Exp Eye Res 32:451, Whikehart DR and Fletcher RT: Cyclic nucleotides in rabbit corneal endothelial cells: fresh tissue vs. tissue culture. Exp Eye Res 28:285, Solomon Y, Londos C, and Rodbell M: A highly sensitive adenylate cyclase assay. Anal Biochem 58:541, Walkenbach RJ, LeGrand RD, and Barr RE: Characterization of adenylate cyclase activity in bovine and human corneal epithelium. INVEST OPHTHAL- MOL VIS SCI 19:1080, Friedman DL, Johnson RA, and Zeilig CE: The role of cyclic nucleotides in the cell cycle. Adv Cyclic Nucleotide Res 7:69, Butterfield LC and Neufeld AH: Cyclic nucleotides and mitosis in the rabbit cornea following superior cervical ganglionectomy. Exp Eye Res 25:427, Reversal of anoxic corneal swelling by breathing oxygen. BARRY A. WEISSMAN, IR- VING FATT, AND BARRY HORN. The anterior corneal surface of nine normal human subjects was made anoxicfor 1 to 3 hr, and corneas swelled a mean of 7% by optical pachometry. Subjects then breathed hyperoxic gas for about an hour, and swelling decreased to the 2% level. (INVEST OPHTHALMOL Vis Sci 22: , 1982.) Oxygen insufficiency at the anterior corneal surface causes corneal swelling. 1 ' 2 The exact cause of this change is unclear because corneal hydration is thought to be primarily controlled by the activity of the endothelial layer, 3 5 which should always have an adequate oxygen supply from the adjacent aqueous humor. 6 In experiments where the subject breathes hyperoxic gas mixture, the increased oxygen tension of the aqueous humor does not give a corresponding increase of oxygen tension at the anterior corneal surface or a change in oxygen flux at this surface. 7 Such experiments suggest that the increase in oxygen tension presumed to occur at the aqueous humor-endothelium boundary 8 " 10 does not cause oxygen to reach the anterior surface of the cornea. Nonetheless, experiments described here demonstrate corneal deswelling when subjects breathe hyperoxic gas after corneas have been swollen by epithelial surface anoxia. Methods. Epithelial surface oxygen insufficiency was produced in one eye of each of nine human subjects by either passing humidified nitrogen gas across a cornea under a tight diver's goggle (after the method of Poise and Mandell 2 ), or by fitting a polymethylmethacrylate contact lens approximately 1 diopter steeper than the subject cornea (steep fit defined by minimal tear exchange, confirmed by fluorescein pooling and stasis). All subjects were young adult volunteers (ages 23 to 41 years, mean 27.8 years). The goggle or lens was removed briefly for measurement. Anoxic conditions at the anterior corneal surface were maintained from 1 to 3 hr. Subjects then breathed hyperoxic gas of approximately 730 mm Hg oxygen tension (breathing system and tests have been previously described 7 ) for 40 to 60 min. We have previously demonstrated 7 that oxygen tension increases in both exhaled gas and the palpebral conjunctival circulatory system under these conditions. One eye served as a control for the swelling part of the experiment, since only one eye was exposed to both an anoxic external and hyperoxic internal environment, and both eyes were monitored for thickness changes. In several experiments, breathing of hyperoxic gas was discontinued but epithelial surface anoxia was maintained. In three experiments (control), all equipment was connected but no hyperoxic gas was breathed. Central corneal thickness was measured periodically by a trained observer throughout all experiments. Care was taken to ensure that these measurements were not biased. A Haag-Streit Pachometer Model I was mounted on a Marco biomicroscope/slit lamp modified for improved central corneal measurement by the technique of Hedbys and Mishima." Calibration was by three polymethylmethacrylate shells of known thickness. Five readings were taken for each measurement: mean of the standard deviation was about /82/ $00.40/ Assoc. for Res. in Vis. and Ophthal., Inc.

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

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