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Volume 18 Number 8 Reports 867 of a primary infection, although examination of rats in other experiments at this same time of infection has shown an increase in intestinal1' 6 and lung7 mast cells. These observations suggest that the stimulus to mast cell proliferation present during infection does not involve the eyes; possibly the stimulating factor is only elaborated by tissues that are in direct contact with larval or worm products. by measuring the degree of inhibition produced by acetazolamide at two standard concentrations. Also, the effect of incubating with iodoacetate was determined in two pairs of these eyes. Only isoenzyme C was detected. Accordingly, it seems that differences in reduction of intraocular pressure that are common among patients treated for glaucoma with systemic carbonic anhydrase inhibitors, despite uniform serum concentrations, are not attributable to interindioidual variation of carbonic anhydrase isoenzymes in the ciliary processes. From the Departments of Ophthalmology and Medicine, Harvard Medical School, the Department of Cornea Research, Eye Research Institute of Retina Foundation, and the Clinical Immunology and Allergy Units, Medical Services, Massachusetts General Hospital, Boston, Mass. This work was supported by grants EY-02099 and AM-20580 from the National Institutes of Health, a grant from Bausch & Lomb, and grants from the National and Massachusetts Chapters of the Arthritis Foundation. Submitted for publication Dec. 4, 1978. Reprint requests: Mathea R. Allansmith, M.D., 20 Staniford St., Boston, Mass. 02114. Recent chronic dose-response studies of carbonic anhydrase inhibitors (CAIs) in patients with glaucoma have indicated that among glaucoma patients there is marked variation from person to person in the influence of CAIs on intraocular pressure, despite similar dosages and serum levels of the medications.1"2 Certain patients are clearly "hyporesponders," despite high dosage therapy.1 The reason is not known. There are in human red blood cells two carbonic anhydrase (CA) isoenzymes, B and C. Acetazolamide is a much more effective inhibitor of isoenzyme C than of B. If there were differences in isoenzymes in tissues that form aqueous humor in different individuals, this could help explain the variable interindividual effect of CAI therapy. Accordingly, we thought it important to examine the type of CA isoenzymes present in a series of human ciliary bodies. Materials and methods. Human eyes enucleated post-mortem were stored at 4° C in a moist chamber and dissected within 24 hr. The ciliary processes, which are assumed to be the principal portion secreting aqueous humor, were identified and excised from 10 pairs of human eyes. The tissue from each pair was centrifuged with 5 ml of distilled water for 5 min at 900 x g in a precooled MSE bench-top centrifuge. The resulting pellet was homogenized in 10 times the weight of distilled water and centrifuged in a Sorvall RC-2B refrigerated centrifuge at 27K x g for 10 min. The supernatant was retained and kept at 4° C, to be assayed for CA activity. CA activity of each sample was determined by the "micro-method" of Maren.3 The amount of tissue required to provide 0.5 enzyme unit of activity was determined. The isoenzyme distribution in each sample was determined by measuring the degree to which enzyme activity was inhibited by 25 and 50 ng of acetazolamide, which differentially inhibits isoenzymes B and C.4 Standard curves were established each day with pure isoenzymes obtained from Sigma Chemical Co., St. Louis, Mo. Human pairs 1 to 4 were studied on day 1, and human pairs 5 to 10 on day 2. Key words: histamine, immunology, anaphylaxis, mast cells, allergy, inflammatory mediators REFERENCES 1. Miller, H.R.: Immune reactions in mucous membranes. II. The differentiation of intestinal mast cells during helminth expulsion in the rat, Lab. Invest. 24:339, 1971. 2. Bloch, K.J., and Wilson, R.J.: Homocytotropic antibody response in the rat infected with the nematode Nippostrongylus brasiliensis. III. Characteristics of the antibody, J. Immunol. 100:629, 1968. 3. Wilson, R.J., and Bloch, K.J.: Homocytotropic antibody response in rat infected with the nematode, Nippostrongylus brasiliensis. II. Characteristics of the immune response. J. Immunol. 100:622, 1968. 4. Mallory, F. B.: Unna's method for mast cells. In Pathological Technique. New York, 1961, Hafner Publ. Co., p. 175. 5. Levene, R.Z.: Mast cells and amines in normal ocular tissues, INVEST. OPHTHALMOL. 1:531, 1962. 6. Keller, R., Cottier, H., and Hess M.W.: Mast cell responses in mesenteric lymph nodes to infection of rats with the nematode, Nippostrongylus brasiliensis, Immunology 27:1039, 1974. 7. Wells, P. D.: Nippostrongylus brasiliensis: mast cell populations in rats, Exp. Parasitol. 30:30, 1971. Identification of isoenzyme C as the principal carbonic anhydrase in human ciliary processes. PAULA C. DOBBS, DAVID L. E P STEIN, AND P. JOHN ANDERSON. Carbonic anhydrase was extracted from the excised processes of the ciliary bodies of 10 pairs of enucleated human eyes, and the isoenzyme composition was assayed 0146-0404/79/080867+04$00.40/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933087/ on 06/17/2017 868 Invest. Ophthalmol. Visual Sci. August 1979 Reports Table I. Comparison of the reaction times* of CA from 10 pairs of human ciliary processes and pure isoenzymes B and C in the presence of inhibitors Dayl B C 1 2 3 4 Uninhibited 445 ± 10 447 ± 6 447 ± 6 447 ± 12 443 ± 12 450 ± 10 Acetazolamide: 0.141 /xM 0.282 /*M 493 ± 12 530 ± 10 533 ±12 530 ± 10 520 ± 20 523 ± 6 Sample Iodoacetate, 10 miM * Units are minutes x 103 ± S.D. All values are means of 3 determinations. Uncatalyzed times were 652 ± 12 and 637 ± 6 for day 1 and day 2, respectively. 0.55- 053- D D V* 051- • 049- \ 047- A • 045040 042 0.44 048 UNINHIBITED TIME IN MINUTES Fig. 1. Plot of inhibited vs. uninhibited times for CA assay in the presence of acetazolamide. Conditions are as in Table I. For day 1 and day 2, respectively: o, • = carbonic anhydrase from pairs of human ciliary processes; A, A = isoenzyme, B; o, • = isoenzyme C. Also, the isoenzyme composition of two of the pairs of eyes was tested by preincubating for 20 hr in 10~2 M iodoacetate, which inhibits isoenzyme B but not isoenzyme C,4> 5 and then assaying for enzyme activity. Results. Results of the CA activity assay for pure isoenzymes B and C and for the ciliary processes of 10 pairs of human eyes are given in Table I. The acetazolamide data were analyzed by a one-way analysis of variance. 6 The results demonstrated that the human samples, pure B, and pure C do not form a homogeneous population (p < 0.01, day 1; p < 0.05, day 2) as might be expected if the human enzymes were mixtures of B and C in various proportions. Selecting isoenzyme B as the likely "odd man out" and testing against the remainder demonstrated a highly significant difference (p < 0.005, both days). No significant differences were found within the remaining groups. These relations are clearly seen in Fig. 1 where the inhibited times (0.141 JUM acetazolamide) are plotted against the uninhibited times of the reaction. The inhibitor constant Ki was obtained by plotting the reciprocal of the enzyme rate vs. the inhibitor concentration. Kms and CO 2 concentration were taken from Maren et al.4 The mean value of the 10 ciliary process samples was 0.102 ± 0.013 )LtM; for isoenzyme C (mean of day 1 and day 2), 0.093 ± 0.002 fxM; and similarly for isoenzyme B, 0.169 ± 0.013 fxM. Errors are expressed as standard deviations. In two samples, there was enough material left to test the differential inhibition of isoenzyme B and C with iodoacetate. The result (Table I) accords closely with the acetazolamide findings. Discussion. Since, as determined by this method, the CA of human ciliary processes from the eyes of 10 people has been indistinguishable from pure isoenzyme C, it seems that variation in amount of isoenzyme B and C does not account for the observed variation in reduction of intraocular pressure in different patients in response to CAI therapy. Variability in response from person to person has been common, with essentially no response in about one out of six glaucomatous patients. We would expect that a difference in isoen- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933087/ on 06/17/2017 Volume 18 Number 8 Reports 869 Day 2 B C 5 6 7 8 9 10 427 ± 6 423 ± 6 420 ± 17 420 ± 10 423 ± 6 423 ± 12 423 ± 6 413 ± 12 467 ± 15 483 ± 6 507 ± 6 523 ± 6 497 ± 6 530 ± 10 510 ± 17 513 ± 15 497 ± 6 523 ± 6 507 ± 12 520 ± 10 500 ± 17 527 ± 6 497 ± 15 517 ± 12 610 ± 10 420 ± 10 zymes might have shown up at least once among our 10 human control donors if this were a factor. There are other possible factors to consider, which include the following. We do not know whether there are variations from person to person in the total amount of CA present in ciliary processes. There is still the possibility of differences in isoenzymes in glaucoma eyes not present in normal eyes. There may be variations among different patients in the contributions of secretion and ultrafiltration in production of aqueous humor. There may be barriers between the blood supply and the site of CA in the tissue. There may be nonspecific binding of acetazolamide in some ciliary processes, interfering with the action of the drug on the enzyme. There is a possibility that isoenzyme B is present in quantities too small for us to detect, yet may have an influence on aqueous humor secretion. In addition, recent studies have indicated that CA may also be present in the pars plana of the ciliary body. 7 If CA in the pars plana has a role in production of aqueous humor, it will be important to measure the amount of enzyme and its isoenzyme composition in this tissue. We would, of course, like to assay CA isoenzyme in glaucomatous eyes, especially in eyes from patients with clinically determined different degrees of responsiveness to CA inhibitors, but practically it is extremely difficult to get the appropriate tissues. In this study, not enough material was available from single pairs of eyes to carry out a full doseresponse study. This precluded our calculating an I50 for acetazolamide as published by Maren and coworkers.4' 8 However, by making a mechanistic assumption, we were able to calculate a Ki for acetazolamide which should be roughly comparable to an I50. The model chosen was the classic competitive inhibition,9 which yields a straight 427 ± 6 413 ± 6 line when the reciprocal rate is plotted vs. the enzyme concentration. It was evident from our data that the model fitted rather poorly. Given these limitations, the agreement of our Ki value for isoenzyme B and the I50 of Maren et al. 8 is excellent (17 and 25 x 1(T8M, respectively). The agreement for isoenzyme C is less good (9.3 and 1.1 x 10~8M, respectively). Reassuringly, the plot of reciprocal rate vs. inhibitor concentration for pure C closely matches that for the human ciliary body enzyme (slope/intercept = 15 and 16 X 10~8M, respectively). Therefore the Ki for the human enzyme was computed with the Km for pure C. We are grateful for the assistance of Jennifer J. Anderson, who performed the analysis of variance. From the Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Mass. Supported in part by the Abraham Silver Memorial Fund of Fight for Sight, Inc., New York, N. Y., and National Eye Institute research grants RO1 EY01894, RO1 EY00002, and TO1 EY00089. Submitted for publication March 2, 1979. Reprint requests: David L. Epstein, M.D., Howe Laboratory, 243 Charles St., Boston, Mass. 02114. Key words: carbonic anhydrase, ciliary processes, acetazolamide, isoenzymes, aqueous formation REFERENCES 1. Dahlen, K., Epstein, D.L., Grant, W.M., Hutchinson, B.T., Prien, E.L., and Krall, J.M.: A repeated dose-response study of methazolamide in glaucoma, Arch. Ophthalmol. 96:2214, 1978. 2. Berson, F.G., Epstein, D.L., Grant, W.M., Hutchinson, B.T., and Dobbs, P.C.: A dose-response study of acetazolamide sequels and tablets in the treatment of glaucoma, INVEST. OPHTHALMOL. VISUAL SCI. 17(ARVO Suppl.):121, 1978. 3. Maren, T.: A simplified micromethod for the deter- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933087/ on 06/17/2017 870 4. 5. 6. 7. Invest. Ophthalmol. Visual Sci. August 1979 Reports mination of carbonic anhydrase and its inhibitors, J. Pharmacol. Exp. Ther. 130:26, 1960. Maren, T., Rayburn, C , and Liddell, N.: Inhibition by anions of human red cell carbonic anhydrase B: physiological and biochemical implications, Science 191:469, 1976. Coleman, J.: Metal ions and enzymatic catalysis. In Kaiser, E., and Kezdy, F., editors: Progress in Bioorganic Chemistry, New York, 1971, John E. Wiley and Sons, Inc., p. 282. Goldstein, A.: Biostatistics, An Introductory Text, New York, 1964, The MacMillan Co., pp. 63-87. Lutjen-Drecoll, E., Lonnerholm, G., and Ober, M.: Carbonic anhydrase activity in the monkey anterior ocular segment. INVEST. OPHTHALMOL. VISUAL SCI. 17(ARVO Suppl.):162, 1978. 8. Maren, T.H., Haywood, J.R., Chapman, S.K., and Zimmerman, T.J.: The pharmacology of methazolamide in relation to the treatment of glaucoma. INVEST. OPHTHALMOL. VIS. SCI. 16:730, 1977. 9. Walter, C : Steady-State Applications in Enzyme Kinetics. New York, 1965, The Ronald Press Co., pp. 104 et. seq. Aqueous humor dynamics following total iridectomy in the cynomolgus monkey. PAUL L. KAUFMAN. Twenty-two cynomolgus monkeys underwent unilateral total iridectomy in vivo. Several weeks to several months postoperatively, intraocular pressure (IOP) was determined bilaterally by manometry under pentobarbital anesthesia (15 monkeys), by a minified Goldmann applanation tonometer under Cl-744 anesthesia (16 monkeys), and by a minified Draeger applanation tonometer under light phencyclidine catalepsia (4 monkeys). Mean IOP in aniridic eyes was about 0.3 mm Hg, or about 3%, lower than in opposite eyes. lOPs of aniridic and opposite eyes of the individual monkeys were highly correlated in all groups. In 11 monkeys, resting total outflow facility and the facility response to intravenous pilocarpine were determined 6 to 9 weeks postoperatively by two-level constant-pressure perfusion under pentobarbital anesthesia. There were no significant differences between mean resting facility, postpilocarpine facility, or facility response to pilocarpine of aniridic and opposite eyes. Resting facility, postpilocarpine facility, and facility response to pilocarpine in aniridic and opposite eyes of the individual monkeys were each highly correlated. Total iridectomy in the cynomolgus monkey apparently has little, if any, effect on IOP, outflow facility, or facility response to intravenous pilocarpine, and the iris plays little, if any, role in mediating the facility response to intravenous pilocarpine. In vervet monkey (Cercopithecus ethiops) and cynomolgus monkey (Macaca fasdcularis), the entire iris may be removed at its root via a small limbal incision in vivo.1 The anterior segments of surgically aniridic eyes, including trabecular meshwork, Schlemm's canal, and ciliary body, appear normal by biomicroscopic, gonioscopic, and light and electron microscopic examination.1' 2 Physiologically, these eyes exhibit, under pentobarbital anesthesia, resting intraocular pressures, 3 outflow facilities,3"5 and facility responses to intravenous and intracameral pilocarpine 3 and to intracameral cytochalasin B 5 which are within the range seen in normal eyes of these species. Nonetheless, since surgically aniridic monkey eyes are used in experimental studies of aqueous humor outflow, ciliary body physiology, and druginduced cataractogenesis, it seemed important to more rigorously define the effect of total iridectomy on aqueous humor dynamics. Materials and methods. Twenty-two adolescent and adult cynomolgus monkeys of both sexes, weighing 1.6 to 4.9 kg, were studied. Each monkey underwent total iridectomy1 in one eye; the opposite eye was untouched. Intraocular pressure (IOP) studies. The monkeys were grouped according to type of anesthesia and method of IOP measurement. Group 1 consisted of 13 monkeys, seven with the right eye aniridic and six with the left eye aniridic. Five weeks postoperatively, the monkeys were anesthetized with intramuscular CI-744, 5 mg/kg, 6 and placed prone in a head holder. IOP was measured in both eyes (right first) with a slit lamp-mounted Goldmann applanation tonometer minified and calibrated manometrically for cynomolgus eyes (P. L. K., unpublished). Group II consisted of three monkeys, all with aniridic right eyes. Both eyes of these monkeys had undergone two-level constant-pressure perfusion of the anterior chamber 6 weeks postoperatively to determine outflow facility.3' 7 Ten weeks postoperatively and 4 weeks after anterior chamber perfusion, the monkeys were anesthetized, and IOP was determined as in Group. I. Group HI consisted of the 16 monkeys of Groups I and II combined. Group IVa consisted of four monkeys, three with aniridic right eyes and one with an aniridic left eye. Both eyes of these monkeys had undergone anterior chamber perfusion at 1- to 2-month intervals postoperatively; at each perfusion, both eyes received the same dose of the same drug intracamerally. Five weeks after the last perfusion, and 7.5 to 9.5 months postoperatively, the monkeys were tranquilized with intramuscular phencyclidine HC1, 0.6 mg/kg, and held upright by an assistant. One drop of 0.4% benoxinate HC1 was 0146-0404/79/080870+06300.60/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc. 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