Ciliary Muscle Muscarinic Binding Sites, Choline Acetyltransferase, and Acefylcholinesterase in Aging Rhesus Monkeys

B'Ann True Gobelr,* Paul L. Kaufman,* and Jon PV Polanskyf

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and the affinity and number of muscarinic binding sites (as reflected by specific 3H-quinuclidinyl benzilate binding) were determined in the ciliary muscle of rhesus monkeys ranging in age from 1-34 years. No age dependence was evident for any of these parameters. Within the limits of their specificity and precision, the data indicate that biochemical alterations in ciliary neuromuscular mechanisms do not account for the age-related loss of ciliary muscle configurational responses to topical pilocarpine and electrical stimulation or the Edinger-Westphal nucleus in the rhesus monkey. Invest Ophthalmol Vis Sci 31:2431- 2436,1990

Presbyopia, the age-related decline in accommoda- line-synthesizing (choline acetyltransferase; ChAT; tive amplitude, occurs seemingly invariably in E.C.2.3.1.6) and -degradating (acetyl cholinesterase; humans,1"3 and has more recently been found to AChE; E.C.3.1.1.7) enzymes, and the muscarinic occur in rhesus monkeys along a comparably relative binding site content and affinity for quinuclidinyl time course.4"8 The etiology and pathophysiology of benzilate (QNB) in rhesus monkeys ranging in age presbyopia in both humans and rhesus monkeys is from 1-34 yr. unknown.5'910 Early studies emphasized lenticular factors and discounted the involvement of the ciliary Materials and Methods muscle.""17 However, it has recently been shown that in the Animals and Eyes rhesus monkey there is an age-related decrease in the magnitude of ciliary muscle excursion in response to Ciliary muscle tissues from 54 rhesus monkeys electrical stimulation of the Edinger-Westphal nu- 718 19 (Macaca mulatto) of both sexes, ranging in age from cleus or to topical pilocarpine, and age-related 1-34 yr, were studied (Table 1); 38 were used in re- structural changes in the ciliary muscle and 19 20 ceptor-binding studies, and 35 were used in enzyme nerves. ' These findings raise the possibility of cili- determinations. Animal ages were based on birth and ary neuromuscular involvement in the pathophysiol- acquisition records from the Wisconsin Regional Pri- ogy of presbyopia. mate Research Center (WRPRC). The ages of the We, therefore, examined the age-dependence of animals born at WRPRC were accurate. The ages of several cholinergically related biochemical parame- those acquired by WRPRC are accurate to within 2 ters of the parasympathetically innervated ciliary yr, since those animals and their records were ob- smooth muscle of the rhesus monkey. Specifically, tained from known reliable sources or compared with we measured the Km values and levels of acetylcho- growth charts when the animals were young and healthy. Eyes were obtained immediately before or at the time of death from animals which were killed due to illness, decline due to old age, or as part of other From the *Department of Ophthalmology, University of Wis- consin-Madison, Madison, Wisconsin, and the fDepartment of protocols. The eyes were immediately placed on ice Ophthalmology, University of California, San Francisco, Califor- and were processed within 30 min as previously de- nia. scribed.21 Some eyes had been totally iridectomized Supported by grants from NIH (EY02698, EY04146, EYO398O, at least 3 months before enucleation, and some had and RR00167) and from the American Health Assistance Founda- received pilocarpine or carbachol on one to three oc- tion, Rockville, Maryland. Reprint requests: Paul L. Kaufman, MD, Department of Oph- casions. thalmology, University of Wisconsin-Madison, Clinical Science These studies conformed to the ARVO Resolution Center, 600 Highland Avenue, Madison, WI 53792. on the Use of Animals in Research.

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Table 1. Experimental tissue

Monkey Age Last cholinergic drug no. (yr) Sex treatment Iridectomy Eye/assay

83130 1 M OD/-14dC OU OS/R 84113 1 F None OU/E 84037 1 M None OD/R, OS/E 83059 2 M OD/-5 m C OU OD/R 82077 3 F None OS/E 82051 3 M None OD/E, R Rhl 3.5 M None OD/R Rh3 3.5 M None OU/R 80102 3.5 F None OS/R 82049 4 F None OS/E, R 20422 5 M None OD/R, OS/E A160 6 M None OS/E, R 79116 6 F None OD/E 79150 8 F None OD/E, OS/R 78012 8 F None OS/E AG88 9 M OD/-19 m C, OS/-20 m C OU OD/E, R AF98 10 F None OS/E AF94 10 F None OS/E AC25 11 F None OU/E, OD/R AC02 11 F None OD/E AC39 12 F None OS/E, R AA20 13 F None OS/E X94 13 F OU/-3 y C OS OD/E, R Y87 14 F None OD/R R70 15 F None OS/E 172 16.5 F OS/-4 y P OS/E 1719 18 F None OS/E, R 1509 20 F None OU OD/R, OS/E 879 20 F None OU OD/E 1457 21 F None OU/R 1519 22 M OS/-18 mC OD/R B66 24 M OS/-4 m C OS/R 1280 24 F OU/-16mC OD OS/E 1409 25 F None OU/R 1421 25 F OD/-4 y C, OS/-4 y P OU/R 1279 25 F OD/-17 mC OD/E PC331 25 M OS/-6 m C OS/R 562 25 F None OS/R 1518 >25 M None OD/R, OS/E 1517 26 M None OU/R 1507 26 M None OS OS/E 649 27 F OU/-15mC OD/E, R 1250 27 F None OS/E, R 068 27 F OD/-1 mC OU/R, OS/E PP27 29 F None OD OS/E, R 789 30 F OD/-2 to 3 h C OD/R 1589 30 F None OS/E O738 31 F None OD/E, R O32 31 F OD/-3 y C OD/R, OS/E O536 33 F None OS/R 342 34 M OS/-4 y C, OD/-3 y C OD/R, OS/E 368 34 M OD/-2 to 3 h C OD/R, OS/E OR68 34 M OD/-14dC OS/R OR15 34 F OD/-4 d C OS/R

C = carbachol; P = pilocarpine; h = hours; d = days; m = months; y assay; OD = right eye; OS = left eye; OU = both eyes. • years before enucleation; R = muscarinic receptor assay; E = enzyme

3H-QNB Assay for Muscarinic Binding Sites groups by 3H-QNB (New England Nuclear, Boston, MA, or Amersham, Arlington Heights, IL) binding 21 Muscarinic receptor number (Bmax) and affinity according to published methods. This ligand appar- (Kd) were determined by two different laboratories ently binds with high potency and similar affinity to 22 23 (Madison-Bmax and K^; San Francisco-Bmax) on dif- all known muscarinic receptor subtypes. ' The ferent samples from different animals of all age protocol for samples run in Madison was altered

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slightly in that samples were incubated at 37°C for 1 based on the relative species life expectancies.933 The hr and collected on glass-fiber filters using the Ska- Km and Vmax values for ChAT and AChE were not tron cell harvester (Sterling, VA).24-25 Both K<, and significantly different in the three age groups (Table Bmax at the Madison location were determined from 2) and were not correlated with age (Fig. 1). There Scatchard plots.26 were also no differences between sexes. The average Vmax/mg values Oumol/min/mg protein) for all ani- Assay for ChAT and AChE mals (n = 35) were: ChAT = 2.74 ± 0.15 X 10"4 and 3 AChE = 1.37 ± 0.08. The Km (MM) values for all The ChAT activity was measured using H-acetyl animals (n = 18) averaged: ChAT = 0.024 ± 0.002 coenzyme A (0.01 ixM) (New England Nuclear) by and AChE = 69.9 ± 4.3. the method of Rand and Johnson27 as described by Binding-site affinity and number did not differ sig- Erickson-Lamy et al28 with the following modifica- nificantly in the three age groups (Table 3) and were tions: choline concentration was adjusted to 0.3 raM; not correlated with age (Fig. 2). There was again no NaCl concentration was adjusted to 60 mM; tetra- difference with respect to sex and no difference in phenyl boron was dissolved in acetonitrile (Sigma, St. binding site number (pmol/mg protein) determined Louis, MO); and toluene scintillation cocktail con- in San Francisco (2.73 ± 0.29; n = 16) compared with tained 10% acetonitrile in place of butanol. The Madison (2.67 ± 0.16; n = 22). The average binding AChE activity was assayed using 3H-acetylcholine io- site number (pmol/mg protein) for all animals (n dide (4 IAM) (New England Nuclear) according to = 38) was 2.70 ± 0.15, and the affinity for QNB (pM) Johnson and Russell,29 again as described by Erick- was 85.3 ± 8.8 (n = 22). son-Lamy et al,28 using the modified scintillation cocktail. Vials used in the ChAT assay were silicon- Discussion ized before use.30 Heat-inactivated tissue blanks were included in some determinations for both assays and The classic view of human presbyopia relates the 10~5 M of the specific AChE inhibitor l:5-bis-(4-al- loss of voluntary accommodative amplitude with age lyldi-methyl-ammonium phenyl)-pentane-3-one to decreased deformability and increased size of the diiodide (BW284c51; Burroughs Wellcome, Re- lens or changes in physical properties or geometry of search Triangle Park, NC31) was included as a blank the zonule, with no appreciable loss of ciliary muscle in the AChE assays. Neither of these blanks produced contractility.210'11'1314'16-34'35 However, the underlying results different from that obtained with dilution studies used indirect methods, such as impedance cy- buffer in place of enzyme. clography or in vitro passive stretch and lens defor- The Km for ChAT and AChE was determined as mation, to derive inferences about ciliary muscle the negative slope in Eadie-Hofstee plots (Woolf-Au- function. Although technically innovative, they did gustinsson-Hofstee plots) of velocity versus velocity/ not generate direct, specific information about ciliary substrate concentration. The fractional conversion of muscle topography, structure, and contractility. substrate to product was converted to Vmax values using the initial rate approximation of the Michaelis- Menton equation.32 Table 2. ChAT and AChE in rhesus monkey ciliary muscle

Statistical Analysis Age (yr)

Grouped data for Y^, Bmax, Km, and Vmax were ex- 1-10 11-20 21-34 pressed as the arithmetic mean ± one standard error of the mean (SEM). Comparisons between groups ChAT* 2.30 ± 0.26 2.02 ± 0.28 2.82 ±0.37 were made by the two-tailed, two-sample student t- AChE 6.80 ±0.91 6.43 ±0.61 7.61 ±0.20 test for unpaired data. Continuous age versus param- n = 8 n = 4 n = 6 eter relationships were assessed by least-squares linear ChAT* 2.59 ± 0.27 2.67 ±0.31 2.97 ±0.21 regression, and the resulting correlation coefficients AChE 1.34 ± 0.15 1.42 ±0.17 1.35 ±0.08 were tested against 0. n= 13 n = 10 n= 12

Data are mean ± SEM for n animals, each contributing one eye or the 2 Results average of both eyes. Km is expressed as tM AcCoA X 10 for ChAT or jiM acetylcholine X 10"' for AChE. Vmax/mg is expressed as nmo\ acetylcholine produced X 104/min/mg protein for ChAT or /imol acetate produced/min/ Data were separated into three age groups: 1-10, mg protein for AChE. There were no significant differences between age groups for any parameters. 11-20, and 21-34 yr, corresponding roughly to * Actual values are obtained by multiplying the listed numbers by the 2 4 human age groups of 1-25, 25-50, and 50-75 yr, following factors: ChAT Km X \0~ ; AChE K.m X 10'; ChAT Vm/mg X lO" .

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5.00exp-4

4.00exp-4 < 0.03- max/nr

3.00exp-4

0.02" prot' 2.00exp-4 Fig. 1. Km (left panels) and Vmax/mg (right panels) for 0.01 l.00exp-4 ChAT (top panels) and AChE AChE AChE (bottom panels) as a function 100- r = 0.18 r = 0.04 of age in rhesus monkey cili- n = 18 n=35 ary muscle. No parameter -2 80- nan correlates with age. n S*L 60 Do a 40 DI

10 20 30 0 10 20 30 40 Age (years)

The rhesus monkey has an age-related decline in Goniovideography shows that a circumlenticular the maximum accommodative amplitude inducible space is always present in the old animals, so external by topical cholinomimetic drugs4 or midbrain elec- restriction of muscle movement by the lens is un- trical stimulation.7-8 Ciliary muscle movement in re- likely.8 Therefore, we investigated biochemical pa- sponse to cholinergic drugs19 or central stimula- rameters related to cholinergic innervation of the cili- tion7'818 similarly declines with age. The time course ary muscle. relative to lifespan for both phenomena is compara- Both Km and Vmax/mg for ChAT and AChE, the ble to the development of human presbyopia.9 Given primary biosynthetic and biodegradative enzymes for the anatomic and functional similarities of the rhesus 4 7 8 19 36 37 and human accommodative apparatus, - ' ' ' - this 200 constellation of findings suggests that the rhesus r = 0.13 monkey is an excellent animal model for human n = 22 presbyopia and that attention should be redirected 150" o toward studying the pathophysiology involving the ciliary muscle. Q. 100- Age-related alterations in rhesus ciliary neuromuscular structure are seen by light and electron microscopy, but they appear relatively minor, and the mus- 50- cle, in even the oldest animals, does not look dener- o o vated, degenerated, fibrosed, hyalinized, or otherwise so unhealthy so as to be incapable of contracting.19-20

Table 3. Muscarinic receptor affinity and number o in rhesus monkey ciliary muscle t_

Age (yr) o 1-10 11-20 21-34 E

Kd* 9.55 ± 2.48 8.34 ± 1.54 8.03 ± 1.03 n = 6 n = 6 n = 10 E Bmax 2.81 ±0.34 2.51 ±0.33 2.70 ±0.18 n = 12 n = 6 n = 20 10 20 30 Age (years) Data are mean ± SEM pM X 10"' (KJ or pmol/mg protein (Bmas) for n animals, each contributing one eye or the average of both eyes. There were Fig. 2. Receptor affinity (top) and number (bottom), determined no significant differences between age groups for either parameter. 3 * Actual values are obtained by multiplying the indicated numbers for K,, with H-QNB, as a function of age. Neither parameter correlates by 10'. with age.

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the cholinergic neurotransmitter acetylcholine, were An animal model for presbyopia. Invest Ophthalmol Vis Sci the same in ciliary muscles encompassing the entire 23:23, 1982. 5. Kaufman PL, Bito LZ, and DeRousseau CJ: The development species life span. Similarly, the affinity and concen- of presbyopia in primates. Trans Ophthalmol Soc UK 102:323, tration of muscarinic receptors in the ciliary muscle, 1983. as determined by specific QNB binding, did not 6. Crawford K, True B'A, Kaufman PL, and Bito LZ: Effects of change with age. However, since the ciliary muscle various anesthetic and autonomic drugs on refraction and ac- has one of the highest tissue concentrations of musca- commodation in monkeys. ARVO Abstracts. Invest Ophthal- rinic receptors2223'3839 and since QNB binds to all mol Vis Sci 27(Suppl):355, 1986. 22 23 7. Neider MW, Crawford K, True B, Kaufman PL, and Bito LZ: muscarinic receptor subtypes, ' we would not have Functional studies of accommodation and presbyopia in detected age-related differences in their relative pro- rhesus monkeys. ARVO Abstracts. Invest Ophthalmol Vis Sci portions. We did not investigate the effect of age on 27(Suppl):81, 1986. spare receptors (which may be present in young 8. Neider MW, Crawford K, Kaufman PL, and Bito LZ: In vivo monkey ciliary muscle40), signal transduction, second videography of the rhesus monkey accommodative apparatus: Age-related loss of ciliary muscle response to central stimula- messenger linkage and function, and neurotransmit- tion. Arch Ophthalmol 108:69, 1990. ter release, any of which could theoretically contrib- 9. Bito LZ and Miranda OC: Presbyopia: The need for a closer ute to an age-related loss of muscle function. None- look. In Presbyopia, Stark L and Obrecht G, editors. New theless, our data strongly suggest that both choliner- York, Professional Press Books, 1987, pp. 411-429. gic neurotransmitter synthesis by the ciliary nerves 10. Koretz JF and Handelman GH: How the human eye focuses. Sci Am 259:92, 1988. and binding by the ciliary muscle in the rhesus mon- 11. Weale RA: Presbyopia. Br J Ophthalmol 46:660, 1962. key remain intact with age. This may not necessarily 12. Bito LZ and Harding CV: Patterns of cellular organization and 41 43 be true of the human iris sphincter. " cell division in the epithelium of the cultured lens. Exp Eye Res The rhesus ciliary muscle assumes the topography 4:146, 1965. 13. Swegmark G: Studies with impedance cyclography on human associated with contraction, regardless of age or the ocular accommodation at different ages. Acta Ophthalmol presence or absence of cholinergic or anticholinergic (Copenh)46:1186, 1969. drugs, if the globe is sectioned equatorially before 14. Fisher RF: Presbyopia and the changes with age in the human formaldehyde-glutaraldehyde fixation.19 Therefore, crystalline lens. J Physiol (Lond) 228:765, 1973. internal "rigidity" of the aged muscle cannot explain 15. Saladin JJ and Stark L: Presbyopia: New evidence from imped- its in vivo immobility. External restriction of muscle ance cyclography supporting the Hess-Gullstrand theory. Vi- sion Res 15:537, 1975. movement by an increasingly inelastic posterior fixa- 16. Fisher RF: The force of contraction of the human ciliary mus- 44 tion (Bruch's membrane or choroid ) could explain cle during accommodation. J Physiol (Lond) 270:51, 1977. all the findings associated with presbyopia in the 17. Farnsworth PN and Shyne SE: Anterior zonular shifts with rhesus and, by extension, in the human. age. Exp Eye Res 28:291, 1979. 18. 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