Muscarinic Effects on Cellular Functions in Cultured Human Ciliary Muscle Cells

Shun Matsumoto, * Thomas Yorio, * Louis DeSantis,] and Iok-Hou

Purpose. To characterize the pharmacology of the carbachol-induced changes of phospholipase C (PLC) activity and intracellular calcium concentration ([Ca2+]j) in cultured human ciliary muscle cells. Methods. Changes in PLC activity of cultured human ciliary muscle cells were determined by production of inositol phosphates. Single-cell dynamic fluorescence ratio imaging was used to determine [Ca2+];. Results. Carbachol, oxotremorine-M, aceclidine, and pilocarpine stimulated PLC with mean ECsoS of 20, 8, 17, and 2 (JM, respectively. The effect of carbachol on PLC was partially suppressed by extracellular Ca2+ depletion. This muscarinic effect was blocked by muscarinic antagonists, such as atropine (apparent pKj = 9.12, nonselective for muscarinic receptor subtypes), pirenzepine (pKj = 6.76, selective for the Mi receptor subtype), 4DAMP (pKi = 9.25, selective for the M, and M3 subtypes), and fHHSiD (pK< = 7.77, selective for the M3 subtype). In [Ca2+]j experiments, carbachol increased [Ca2+]i transients in human ciliary muscle cells in a dose-dependent manner with a mean EC50 of 7 fjM. 4DAMP was approximately 100 times more potent than pirenzepine in the inhibition of the carbachol-induced [Ca2+]j increase. [Ca2+]j oscillations were observed after carbachol stimulation and persisted after extracellular Ca2+ depletion. Conclusions. Muscarinic agonists activate PLC and increase [Ca2+]i in cultured human ciliary muscle cells through an M3-like muscarinic receptor subtype. Invest Ophthalmol Vis Sci. 1994; 35:3732-3738.

L opical application of muscarinic agonists onto the types, named mi to m5, were discovered, mi, m2, and eye lowers intraocular pressure, presumably because m3 correspond to, respectively, the Mb M2, and M3 of the activation of muscarinic acetylcholine receptors subtypes defined by affinity profiles of selective musca- (mAChR), which in turn induce contraction of ciliary rinic antagonists.5 Because both pilocarpine and car- muscles and increase aqueous humor outflow.12 How- bachol are nonselective among the five receptor sub- ever, in addition to their action on aqueous dynamics, types, it is not clear which receptor subtype or subtypes muscarinic agonists, such as carbachol and pilocar- are responsible for the ocular actions of the muscarin- pine, also have ocular side effects. Their common ad- ics. Therefore, it is possible that their ocular hypoten- verse effects include miosis (due to the contraction of sive effects and their side effects are mediated by dif- the iris sphincter muscle), accommodation (change ferent subtypes and, consequently, that appropriate of lens curvature secondary to ciliary muscle contrac- subtype-specific agonists theoretically could lower in- tion), and browache (supposedly due to severe con- traocular pressure with reduced ocular side effects. traction or spasm of the ciliary muscles) .3>4 Indeed, current evidence indicates that one can sepa- Recently, messenger RNAs of five mAChR sub- rate the therapeutic from the untoward effects of the muscarinics. Aceclidine, another muscarinic agonist, is equipotent to pilocarpine in lowering intraocular From the *Department of Pharmacology, North Texas Eye Research Institute, pressure in human subjects, but it is more potent as University of North Texas Health Science Center at Fort Wmth, and ^Glaucoma 6 9 Research, Alcon Laboratories, Fort Wmth, Texas. a miotic and does not cause much accommodation. " Supported in part by a grant from Alcon Laboratories. In monkeys, aceclidine similarly increases the outflow Submitted for publication September 3, 1993; revised February 17, 1994; accepted April 25, 1994. facility of aqueous humor with minimal accommoda- Proprietary interest category: E, C5. tive changes.10 Additionally, the pilocarpine-induced Reprint requests: Shim Matsumoto, M.D., Department of Ophthalmology, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunhyo-ku, Tokyo 113, Japan. increase in outflow facility in the monkey can be re-

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versed by atropine in two phases, a fast and a slow dium was removed and each well was rinsed with 4X1 phase, whereas the reversal of accommodation shows ml DMEM:nutrient mixture F12 (1:1), supplemented only the fast phase.11 This dissociation of the musca- with 10 mM LiCl (Mallinckrodt, Paris, KY) at room rinic effects agrees with the hypothesis that different temperature. In some experiments, Ca2+ in the me- effects are mediated by different receptor subtypes. dium was chelated with additional ethyleneglycol bis An initial step to test this hypothesis is to identify (2-aminoethylether) tetraacetic acid (EGTA; Sigma, St the receptor subtypes located in the different ocular Louis, MO), forming an EGTA-Ca2+ buffer system (pH tissues. Several published studies attempted to address = 7.4) .20 The cells were incubated with 1 ml of the this issue using receptor binding or techniques of mo- same solution for 10 minutes before 10 (A of agonists lecular biology. In the human iris sphincter, the m3/ were added to the indicated wells. Alternatively, antag- M3 subtype was shown to be the most prominent onists were added at this time and 10 fi\ of carbachol 12 14 mAChR. " The messenger RNA of the m3 receptor (final concentration = 100 /iM) was added 12 minutes was also detected in cultured human ciliary muscle later. After an incubation period of 1 hour, the assay cells and in postmortem tissues.15"17 Recently, based was stopped by the removal of the medium, and 0.5 on results from receptor binding assays, WoldeMussie ml of perchloric acid (Baker, Phillipsburg, NJ) was 12 et al reported that an M3-like mAChR subtype is pres- added. Samples were then incubated on ice for 10 ent in cultured human ciliary muscle cells, and it was minutes. The perchloric acid was later removed by suggested that this receptor may be involved in the extraction with 2 ml of 1:1 (vol/vol) 1,1,2-trichloro- carbachol-induced activation of phosphoinositide me- trifluoro-ethane:tri-n-octylamine (Sigma) mixture. tabolism. Because studies that use ligand binding or The resulting aqueous layer was loaded onto anion- Northern blot analysis can only demonstrate the pres- exchange columns (AG 1-X8 anion exchange resin ence and the cellular concentration of the receptor in formate form, 1 ml; BioRad, Hercules, CA). The protein and the messenger RNA, they do not directly columns were washed with 10 ml of water and 7 ml define the relative importance of the receptor sub- of 50-mM ammonium formate (Sigma). The bound types in cell functions. To expand these studies fur- inositol phosphates were then eluted with 2X2 ml ther, we used cell functional assays (phospholipase C of 1.2 M ammonium formate-0.1 M formic acid activity and intracellular calcium concentration) to try (Sigma). The radioactivity in the eluates was counted to elucidate the functional involvement of muscarinic with a /3-scintillation counter. In recent experiments, receptor subtypes in the cultured human ciliary mus- the assay procedures were simplified such that the cle cells. reaction was terminated by replacing the cell medium with 1 ml of 0.1-M formic acid instead of perchloric acid. The cell lysate was then loaded directly onto the MATERIALS AND METHODS anion-exchange column and eluted, and radioactivity was measured as described. The two assay procedures Cells generated similar results (data not shown). PLC activ- The human ciliary muscle cell strain established by ity of the cells was estimated from the accumulation Tamm et al18 was used. The cells were cultured at of radioactive inositol phosphates. 37°C in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY), supplemented with 10% fetal calf Intracellular [Ca2+] Measurement serum (Hyclone, Logan, UT), 4 mM L-glutamine Human ciliary muscle cells were grown on coverslips (Gibco), and 50 Atg/ml gentamicin (Gibco) in humidi- (#0; Biophysica Technologies, Baltimore, MD) in 6- fied air containing 5% CO2. Upon confluence, the well plates (Costar). The culture medium was changed cells were subcultured by trypsinization using 0.05% to serum-free DMEM 24 hours before the calcium con- trypsin-0.53 mM ethylenediamine-tetraacetic acid centration measurement. On the day of the measure- (Gibco). Cells of passages 10 to 14 were used in subse- ment, the cells were incubated for 60 minutes in a quent experiments. HEPES buffer (125 mM NaCl, 5 mM KC1, 1.8 mM CaCl , 2 mM MgCl , 0.5 mM NaH PO , 5 mM Phospholipase C Assay 2 2 2 4 NaHCO3,10 mM HEPES, 10 mM glucose, 0.1% bovine Phospholipase C activity was assayed according to a serum albumin; pH 7.2) containing 5 fiM of a calcium- procedure modified from that described by Downes fluorescent dye, fura-2 acetoxymethyl ester (Fura-2/ and Michell.19 Human ciliary muscle cells grown in AM; Molecular Probes, Eugene, OR). After the incu- 24-well plates (7 X 105 to 8 X 105 cells per well; Costar, bation, the coverslip was rinsed twice with the same Cambridge, MA) were incubated with myo-[2-3H]-ino- HEPES buffer and mounted in a chamber on the stage sitol (specific activity 370 to 740 GBq/mmol; Amers- of a Nikon Diaphot microscope (Tokyo, Japan). The ham, Arlington Heights, IL) at 5 /t/Ci/ml per well in chamber was filled with 3 ml of the HEPES buffer serum-free Dulbecco's modified Eagle's medium and kept at 37°C during the experiment. Intracellular (DMEM) for 2 days. On the day of the assay, the me- fluorescence intensity of 510-nm emission wavelength,

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desensitization of the receptor during this period. For 12 20 1 1 1 1 1 i i convenience, 1 hour was chosen as the incubation

• 100 /J.U carbachol time in the following studies. Carbachol increased c PLC activity in human ciliary muscle cells 11.2 ± 0.9 o o vehicle times (mean ± SEM of 10 duplicated experiments), 15 - _o from a basal level of 439 ± 67 cpm/well to 4633 ± 593 cpm/well, with a mean EC50 of 20.3 /M (log EC50 E = -4.69 ± 0.06, mean ± SEM of 11 duplicated experi- en ments) . Other muscarinic agonists, such as oxotremo- 10 - rine-M, aceclidine, and pilocarpine, stimulated PLC O with EC50s of 7.8 //M (log EC50 = -5.11 ± 0.05, mean CD ± SEM, n = 4), 17.0 fjM (log EC = -4.77 ± 0.09, CO 50 O n = 4), and 2.4 /xM (log EC50 = -5.62 ± 0.10, n = Q. 5 - - 4), respectively (Fig. 2). The maximal stimulation of PLC by oxotremorine-M, a full agonist similar to carba- CL / chol, was 112% ± 4% of that of carbachol. In contrast, If) / O n n the maximal effects of the partial agonists, aceclidine i i _C u i i i and pilocarpine, were only 32% ±1% and 15% ± Q_ 210 40 60 80 100 120 2%, respectively, of that of carbachol. Time (min) Activity of PLC was shown to be calcium dependent.22 The carbachol-induced activation of PLC was FIGURE l. Representative time course of 100-^M carbachol- also dependent on the availability of calcium. When induced PLC stimulation in human ciliary muscle cells. Each symbol represents datum from a single sample. the extracellular concentration of free calcium was decreased from 1.05 mM to 100 fjM, the PLC activation by carbachol was reduced to 35% (Table 1). How- excited by alternating 340 and 380 nm excitation wave- ever, further decrease in the extracellular free calcium lengths, was measured by a dynamic, single-cell video- concentration to levels as low as 10 nM did not cause imaging technique using Image-1/FL Quantitative a further reduction of the carbachol effect. Changes Fluorescence System (Universal Imaging, West Ches- in extracellular calcium concentrations did not affect ter, PA). The intracellular Ca2+ concentration basal levels of inositol phosphates. ([Ca2+]j) was calculated from the intensity ratio of To elucidate the involvement of muscarinic recep- fluorescence at these two excitation wavelengths ac- tor subtypes in the activation of PLC by carbachol, cording to the equation of Grynkiewicz et al.21 Various compounds (3 /A each) were added di- ^ 20 i i i r rectly into the chamber as indicated. The chamber O was washed 3 times with the HEPES buffer between • carbachol additions of agonists. V oxotremorine-M Chemicals 15 T aceclidine Carbachol, oxotremorine-M, ( + )-pilocarpine hydro- • pilocarpine chloride, pirenzepine dihydrochloride, p-fluoro

hexahydro-sila-difenidol hydrochloride (fHHSiD), if) and 4-diphenylacetoxy-N-methylpiperidine methio- 10 dide (4DAMP) were purchased from Research Bio- o CD chemicals (Natick, MA). Atropine sulfate was pur- if) chased from Sigma (St. Louis, MO). Aceclidine was O purchased from Alcon Laboratories (Fort Worth, TX). CL ~O SI CL RESULTS o Phospholipase C Activity Q_ -7 -6 -5 -4 -3 The time course of carbachol-induced PLC stimulation is shown in Figure 1. After 100-^iM carbachol log [agonist] (M) treatment, accumulation of inositol phosphates in- FIGURE 2. Representative dose-response curves of muscarinic creased continuously for at least 2 hours, indicating agonists for PLC stimulation in human ciliary muscle cells. no significant depletion of the enzyme substrate or Each symbol represents datum from a single sample.

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TABLE l. Effect of Various Extracellular 10000 Concentrations of Free Calcium on Carbachol (100 fM) -Induced Phospholipase C Activity

Extracellular Free [Ca2+] PLC Activity (%) 1000 Washout Washout O U 1.05 mM 100.0 ± 3.2 100 ^M 35.4 ± 2.2 _D 32.9 ± 2.6 1 ,uM 37.0 ± 2.4 "Q3 100 nM 38.2 ± 2.6 o 100 10 nM 33.0 ± 1.4 D Data represent mean ± SEM of 4 experiments, each in duplicates. 1/zM CCH 5/iM CCH

10 various subtype-selective antagonists were used to 10 20 30 40 50 60 block this effect. Thus, pirenzepine (an M] mAChR Time (min) subtype selective antagonist), 4DAMP (an M! and M3 mAChR selective antagonist), fHHSiD (an M3 mAChR FIGURE 4. A typical trace of carbachol (CCH)-induced selective antagonist), and atropine (a nonselective an- [Ca2+]i change in a single human ciliary muscle cell. Similar tagonist) all antagonized the effect of carbachol in a results were obtained in six other experiments. The cell was dose-dependent and competitive manner (Fig. 3). The rinsed with the HEPES buffer between drug additions as inhibition curves were invariably sigmoidal and fitted indicated by "washout." well with a single-site model of drug interaction (Hill's coefficients for all curves were approximately 1), im- K< = IC /(l + [carbachol]/(EC of carbachol)) plying that only one receptor subtype was essential in 50 50 Potencies of the antagonists were 4DAMP (pKj = mediating the carbachol-induced activation of PLC in 9.25 ± 0.22, n = 6), atropine (pK< = 9.12 ± 0.10, n these cells. The inhibition coefficient (Kj) of each = 6), fHHSiD (pKj = 7.77 ± 0.15, n = 8), and piren- compound can then be calculated with the equation23: zepine (pKj = 6.76 ± 0.05, n = 7). This potency profile of the antagonists indicates that the M3 or an M3-like mAChR subtype mediates the carbachol effect. Intracellular Ca2+ Concentration 100 T pirenzepine" Mean resting [Ca2+]; in human ciliary muscle cells was D fHHSiD 73.4 ± 9.3 nM (n = 40). Carbachol caused a rapid 1£ 80 2+ U • atropine increase in [Ca ]j in these cells, followed by a slow D decline with concentration oscillations (Figs. 4, 5). Re- V 4DAMP 2+ O 60 peated additions of carbachol caused similar [Ca ]j CD increases and did not show signs of desensitization 2+ CO (Fig. 5). Carbachol-induced peak [Ca ]j changes in- D 40 Q. creased in a dose-dependent manner (Fig. 6) with a ~o mean EC50 of 7 fiM (log EC50 = -5.16 ± 0.17, n = sz 9). As in the PLC studies, the rapid rise of [Ca2+]j a. 20 induced by carbachol was only partially suppressed by o the removal of free extracellular calcium, whereas the si [Ca2+]i oscillations during the sustained presence of carbachol was not affected by the availability of extra- -12 -10 -6 -4 -2 cellular calcium (Fig. 7). This carbachol effect on log[antagonist] (M) [Ca2+]; was mediated by mAChRs. A pretreatment of a 1-nM nonselective muscarinic antagonist, atropine, FIGURE 3. Representative inhibition curves for carbachol-in- completely blocked [Ca2+]i increase induced by 1 /JM duced PLC stimulation in human ciliary muscle cells. Vari- carbachol, but it did not block the [Ca2+]; increase ous concentration of antagonists were added 12 minutes induced by 10 fjM carbachol (Fig. 8). An M]-mAChR before the addition of 100 fjM of carbachol. Hill's coeffi- subtype-selective antagonist, pirenzepine, also blocked cients were calculated to be 0.96, 0.94, 0.94, and 0.96 for 2+ atropine, pirenzepine, 4DAMP, and fHHSiD, respectively. carbachol-induced [Ca ]j increase in human ciliary Each symbol represents datum from a single sample. muscle cells. However, pirenzepine's effect on carba-

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10000

1000 O o

100

10MM CCH 10/iM CCH Washout lyuM CCH

10 10 20 30 40 50 Time (min) 2+ Time (min) FIGURE 7. Effect of extracellular Ca on carbachol (CCH)- 2+ 2+ 2+ induced [Ca ]j increase. [Ca ]; oscillation was still observ- FIGURE 5. A typical trace of [Ca ]i change in a single human able in Ca2+-free HEPES buffer (indicated by the bar above ciliary muscle cell after repeated stimulations of 10-//M car- the graph). Similar results were obtained in four other ex- bachol (CCH). No signs of desensitization were observed. periments. Similar results were obtained in four other experiments.

chol-induced [Ca2+]; increase was approximately 100 activated PLC and increased the [Ca2+]i in a dose- and times less potent than that of an Mj- and M3-mAChR time-dependent manner. Muscarinic agonists, such as subtype antagonist, 4DAMP (Fig. 9), suggesting that carbachol and pilocarpine, also cause contraction in 24 the M3 mAChR subtype was essential in mediating the these cells. It is likely, though unproven, that the Ca2+ effect of carbachol. agonist-induced contraction is mediated by the activation of the PLC followed by an increase in [Ca2+]j, DISCUSSION because similar activation-contraction pathways have been described in other smooth muscles.25"27 How- In this report, we have demonstrated that, in the cultured human ciliary muscle cells, muscarinic agonists

1000 -

Washout Woshout Woshout C 1000 Washout woshout Wash0"t Washout

D O

100

CCH 10/iM CCH 1/zM C 100/iM CCH 5/iM CCH 50/iM CCH 1nM At 10nM At

10 10 10 20 30 10 20 30 40 50 Time (min) Time (min) FIGURE 8. Effect of atropine (At) pretreatment on carbachol 2+ FIGURE 6. A typical trace of [Ca ]i change in a single human (C)-induced [Ca2+]i increase. The trace indicates [Ca2+]; ciliary muscle cell with various concentrations of carbachol change in a single human ciliary muscle cell. Atropine (CCH). Responses were dose dependent. Similar results blocked the carbachol effect in a dose-dependent manner. were obtained in eight odier experiments. Similar results were obtained in four other experiments.

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ever, as in other smooth muscles, not all activators of Further studies of these cells are needed to clarify this PLC and [Ca2+]; stimulate contraction. In the ciliary hypothesis. 2+ muscle cells, histamine also increases [Ca ]; and the In the present study, we also demonstrated that accumulation of inositol phosphates, but it did not an M3-like mAChR subtype was essential in mediating seem to cause the cell to contract (unpublished obser- the carbachol effects. Measurements of [Ca2+]i vations, 1993). This divergence of actions of the same showed that 4DAMP, an Mr and M3-selective mAChR second messengers is still not fully understood. It has antagonist, inhibited carbachol-induced [Ca2+]; in- been speculated that the exact spatial and temporal creases with a potency of approximately 100 times that relationships, as well as the potential compartmentiza- of pirenzepine, an Mj-selective mAChR antagonist. tion of the various intracellular components, deter- This also agrees with results in the PLC assays, in which mine the final functional outcome. Therefore, we can- 4DAMP was a more potent antagonist than pirenzep- not exclude the possibility that the various muscarinic ine in blocking the carbachol effects. This potency actions on the cell, though mediated by similar recep- profile clearly indicates the involvement of the M3 or tors, did not have cause-effect relationships. an M3-like mAChR subtype in the carbachol actions. In these cells, the carbachol action on PLC was These observations are consistent with other pub- only partially dependent on the extracellular concen- lished data in which the M3 receptor is the most promi- tration of free calcium, such that a significant portion nent subtype in the human ciliary muscle or the cul- 1215 17 of the enzyme activity remained after the extracellular tured human ciliary muscle cells. " Similar to our 12 calcium was chelated. This finding indicates that the findings, WoldeMussie et al demonstrated that the cell may have heterogeneous PLC isozymes with vari- muscarinic agonist-induced PLC activation was proba- ous degrees of requirement of calcium or, more likely, bly mediated by an M3-like receptor. However, unlike that intracellular calcium stores played a role in main- our findings, the inhibition curve of 4DAMP in their taining the muscarinic action. This latter interpreta- study had a Hill's coefficient significantly less than 1, tion also agrees with the result that the carbachol- indicating that both carbachol and 4DAMP interacted induced increase in calcium transients persisted in with more than one receptor in their cells, or that the calcium-free medium. Interestingly, the absence cooperative interactions among ligands occurred. In of free extracellular calcium did not diminish the car- either case, the estimated affinity coefficient will be bachol-induced [Ca2+]i oscillations in the ciliary mus- skewed. This difference in Hill's coefficients between cle cells. This phenomenon can be explained by the their results and the present results may be explained two-calcium storage-pool model advocated by Ber- by the potential difference in homogeneity of the cell ridge,28 in which model calcium, released from the strains used or, alternatively, the difference in the ex- inositol 1,4,5 triphosphate-sensitive and-insensitive in- pression of mAChRs induced by different culture con- tracellular calcium stores, results in the oscillation. ditions. These intracellular messenger studies obviously have functional implications for the ciliary muscle. The M3 receptor has been shown to be important for C=carbachol muscarinic agonist-generated contraction of ciliary P = pirenzepine 29 D=4DAMP muscle strips from the rhesus monkey. Furthermore, these in vitro findings correlate well with in vivo stud- 000 - ies reported by Gabelt and Kaufman,30 in which aque- Washout Weshout Washout Washout ous humor outflow facility increased, and accommo-

\ \ dation, induced by perfusion of pilocarpine into the ' ft anterior chamber of anesthetized rhesus monkey eyes, \ \ \ was mediated by the M3-mAChR subtype. 100 \ \ l V ' Thus, our results, obtained from in vitro assays in / the cultured cells, agree very well with those obtained .1 / f f t | 10MM C from studies using tissue strips or in vivo models. Col- 1 10/xM C 1/uM C l^M C C 1 l^M C lectively, the M3 mAChR subtype is overwhelmingly lOOnM P p lnM D in indicated to be essential in the functional changes in the eye produced by muscarinic cholinergic agonists. Time (min) It therefore appears that using receptor subtype-selec- FIGURE 9. Effect of pirenzepine (P) or 4DAMP (D) pretreat- tive agonists to separate the IOP-lowering effect from ment of carbachol(C)-induced [Ca2+]i increase. The trace the ocular side effects of muscarinic compounds will indicates [Ca2+]j change in a single human ciliary muscle be theoretically impossible, unless structures other cell. 4DAMP was much more potent than pirenzepine in than the ciliary muscle that express other receptor blocking the carbachol effect. Similar results were obtained subtypes are also involved in the outflow effect of mus- in four other experiments. carinics. Clarification awaits future studies.

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Key Words type and m3 messenger RNA in human ocular structures. Soc Neurosci Abstract. 1991; 17:587. human ciliary muscle cell, intracellular calcium, muscarinic 15. Bogardus AM, Feldmann BJ, WoldeMussie E, Gil DW. agonist, M3-muscarinic receptor subtype, phospholipase C Muscarinic receptor subtypes in human eye. Soc Neu- rosci Abstract. 1991; 17:587. Acknowledgments 16. Erickson-Lamy KA, Chen MC, Hernandez MR. Expres- The authors thank Drs. Elke Luetjen-Drecoll and Ernst sion of muscarinic receptor mRNA in cultured ciliary Tamm for providing the human ciliary muscle cells. They muscle cells. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1991;32:833. also thank Peggy Magnino for technical help. 17. Zhang X, Hernandez MR, Cheng MC, Erickson-Lamy KA. Further characterization of muscarinic receptor References subtype mRNA in the human ciliary muscle. ARVO 1. Kaufman PL. Aqueous humor outflow. Curr Top Eye Abstracts. Invest Ophthalmol Vis Sci. 1992; 33:1200. Res. 1984;4:97-138. 18. 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Muscarinic receptor subtypes: Physiology of Ca indicators with gready improved fluorescence and clinical implications. N Engl J Med. 1989; properties. / Biol Chem. 1985;260:3440-3450. 321:1022-1029. 22. Rhee SG, Choi KD. Regulation of inositol phospho- 6. Lieberman TW, Leopold IH. The use of aceclidine in lipid-specific phospholipase C isozymes. / Biol Chem. the treatment of glaucoma: Its effect on intraocular 1992;267:12393-12396. pressure and facility of aqueous humor outflow as 23. Craig DA. The Cheng-Prusoff relationship: Somediing compared to that of pilocarpine. Am J Ophthalmol. lost in the translation. Trends Pharmacol Sci. 1993; 1967;64:405-415. 14:89-91. 7. Romano JH. Double-blind cross-over comparison of 24. Pang IH, Shade D, Tamm E, DeSantis L. Single-cell aceclidine and pilocarpine in open-angle glaucoma. contraction assay for human ciliary muscle cells: Effect Br J Ophthalmol. 1970;54:510-521. of carbachol. Invest Ophthalmol Vis Sci. 1993; 34:1876- 1879. 8. Fechner PU, Teichman KD, Weyrauch W. Accommo- 25. Fay FS, Shlevin HH, Granger WC, Taylor SR. Aequorin dative effects of aceclidine in the treatment of glau- luminescence during activation of single isolated coma. Am J Ophthalmol. 1975;79:104-106. smooth muscle cells. Nature. 1979;280:506-508. 9. Keren G, Treister G. Effect of aceclidine (+) isomer 26. Williams DA, Fogarty KE, Tsien RY, Fay FS. Calcium and pilocarpine on the intraocular pressure decrease gradients in single smooth muscle cells revealed by and the miosis in glaucomatous eyes: Effect on accom- the digital imaging microscope using Fura-2. Nature. modation in normal eyes of young subjects. Ophthalmo- 1985;318:558-561. logica. 1980; 180:181-187. 27. Grandordy BM, Cuss FM, Sampson AS, Palmer JB, 10. Erickson-Lamy KA, Schroeder A. Dissociation between Barnes PJ. Phosphatidylinositol response to choliner- the effect of aceclidine on outflow facility and accom- gic agonists in airway smooth muscle: Relationship modation. 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