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(Investigative Ophthalmology and Visual Science. 2000;41:1743-1748.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Direct Effects of Muscarinic Agents on the Outflow Pathways in Human Eyes

Kristine A. Erickson1,2,3 and Alison Schroeder1

From the Departments of 1 Ophthalmology and 2 Pharmacology, Boston University School of Medicine; and 3 The New England College of Optometry, Boston, Massachusetts.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. Recent studies demonstrating the presence of muscarinic receptors and contractile-like cells in the trabecular meshwork tissue and/or cell cultures from human eyes suggest the possibility that there may be a direct effect of muscarinic agonists on outflow facility. The present studies were conducted to determine whether muscarinic agonists could change outflow facility in perfused human ocular anterior segments, which lack an intact ciliary muscle.

METHODS. Human eyes were dissected and perfused according to previously described methods. A steady state baseline facility was established for 90 minutes, after which up to four sequential concentrations ranging from 10-9 to 10-3 M of pilocarpine, aceclidine, or carbachol were added to the perfusion medium. In other studies, 10-6 M atropine was perfused alone followed by 10-7 M carbachol with 10-6 M atropine, whereas fellow control eyes received carbachol alone. Outflow facility was measured for 60 minutes after each drug addition. The outflow facility measurement in each eye after drug administration was compared with the baseline measurement.

RESULTS. Outflow facility increased from baseline facility in eyes treated with pilocarpine, aceclidine, or carbachol at lower concentrations (10-9 to 10-6 M) but remained unchanged at higher concentrations (10-4 to 10-2 M). The effects of carbachol at 10-7 M were completely blocked by atropine.

CONCLUSIONS. Muscarinic agonists increase outflow facility in human eyes by a direct stimulation of the outflow tissues in the absence of an intact ciliary muscle. This effect is biphasic, occurring at concentrations of 10-6 M and lower with no effect at higher concentrations.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The ciliary muscle has been shown to be essential in mediating muscarinic agonist–induced changes in outflow facility in the monkey eye in vivo. Kaufman and Bárány elegantly demonstrated that surgical disinsertion of the ciliary muscle in monkeys prevents an acute response to pilocarpine in outflow facility.1 Muscarinic agonists are thought to bind to receptors in the ciliary muscle, causing contraction of the muscle, displacement of the scleral spur, and a widening of the spaces in the trabecular meshwork facilitating aqueous humor flow out of the eye.2 However, recent studies have demonstrated that cells derived from human trabecular meshwork3 and human trabecular meshwork tissue in situ4 5 have muscarinic receptors. Moreover, meshwork cells with contractile properties have been demonstrated histologically6 and physiologically.7 8 9 Collectively, these studies raise the possibility that stimulation of muscarinic receptors in cells located within the outflow pathways may lead to a direct effect on outflow facility in human eyes that is not dependent on the presence of an intact functional ciliary muscle.

The present study was conducted to test the hypothesis that muscarinic agents increase outflow facility by a direct effect on outflow pathway cells in human eyes.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Procurement of Human Eyes
Forty postmortem eyes from 28 donors (average age, 77.0 ± 1.5 years) with no prior history of ocular disease or surgery were obtained from the National Disease Research Interchange (Philadelphia, PA). Eyes were enucleated within 9 hours (average time, 3.7 ± 0.3 hours) of the donors’ deaths and stored refrigerated in a humid saline environment until they were dissected. Within 15.5 hours (average time, 8.9 ± 1.2 hours) of donors’ death, the eyes were bisected at the equator, and the uveal layer was removed from the anterior segment leaving a corneoscleral shell with attached trabecular meshwork and associated scleral outflow tissue. The eyes were then placed in Optisol (Chiron Ophthalmics, Irvine, CA) on ice and shipped via overnight mail. Before perfusion, the eyes were rinsed in sterile Dulbecco’s modified Eagle’s medium (DMEM) containing 50 U/ml penicillin, 50 µg/ml streptomycin, and 5 µg/ml amphotericin B (DMEM + PSA), mounted on a specialized perfusion chamber, and placed in an incubator at 37°C with 5% CO2. Perfusion was carried out at a constant pressure of 15 mm Hg using DMEM + PSA (all obtained from Sigma Chemical, St. Louis, MO), and outflow determinations were made before and after drug exchange according to methods published previously.10

Drugs and Outflow Facility
Stock solutions of pilocarpine and carbachol (Sigma Chemical) and aceclidine (a gift from Merck Sharp et Dohme–Chibret, Riom, France) and their working dilutions were prepared in DMEM + PSA immediately before perfusion.

Dose–response data for pilocarpine, aceclidine, and carbachol were determined as described previously11 using a sequential exchange of up to 4 concentrations of one of the muscarinic agents in one eye (10-9, 10-8, 10-7, 10-6 M), while another eye received an additional 3 concentrations (10-4, 10-3, 10-2 M). Previous studies established that this tissue preparation remains viable for up to 4 days,10 the outflow tissues have a normal morphology (i.e., the meshwork is not collapsed, cells are plentiful, the endothelial layer is intact, and giant vacuoles can be found in the cells lining Schlemm’s Canal10 ), and the baseline outflow facility remains stable for 6 hours or more.10 11

To verify that outflow facility increases could be blocked by atropine, studies were conducted in paired eyes where one eye received 10-6 M atropine for 60 minutes followed by a sequential exchange with 10-7 M carbachol and 10-6 M atropine, and the fellow eye received sequential concentrations (10-9 to 10-6) of carbachol alone. Typically, steady state levels are obtained within the first 30 minutes after initiating the perfusion and within the first 15 minutes after each drug exchange. All data were obtained after the system had reached a steady state. In both dose–response and blocking studies, steady state outflow facility was determined for 90 minutes before drug administration and for 60 minutes after each sequential drug administration. Maximal drug effects with all three cholinergic agents were reached within the first 15 minutes of perfusion measurement and in all cases persisted for the entire measurement period. Therefore, postdrug facilities typically consisted of the average of four measurements and represented a true cumulative effect. In all experiments, drug effects were evaluated in each eye as the ratio between the average postdrug (Cd) and predrug (Co) facility (C). Control eyes were perfused continuously without drug administration to ensure stability of the baseline facility over the experimental period. Statistical analysis consisted of a paired comparison between the individual ratios (Cd/Co; Table 1 ). This method of data analysis normalizes the individual differences in baseline C and has been used extensively in outflow facility experiments.10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Unlike C in calf and monkey eyes, facility of human eyes is well known to be stable for many hours. This is true of in vitro whole human eyes and cultured human anterior segments.11 13 14 16 23 24 Therefore, in the case of human eyes, it is appropriate to compare postdrug and predrug facilities in a single eye.11


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  		Table 1. Effects of Pilocarpine, Aceclidine, and Carbachol on Outflow Facility in the Human Eye In Vitro

 

   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Outflow facility remained unchanged from baseline facility after perfusion with 10-4 to 10-2 M of pilocarpine, aceclidine, or carbachol. In contrast, outflow facility increased in a dose-related fashion after perfusion with concentrations ranging from 10-9 to 10-6 M of each of the three agonists (Table 1 , Figs. 1 2 3 ). For pilocarpine, a maximal increase in outflow facility of 31% was noted at 10-6 M (Table 1 , Fig. 1 ). Similarly, for aceclidine, the maximal increase of 98% was noted at 10-6 M (Table 1 , Fig. 2 ). Finally, the maximal increase for carbachol of 50% occurred at 10-7 M (Table 1 , Fig. 3 ). In these studies, aceclidine appeared to have the greatest efficacy, approximately 2.0 to 2.5 times more than either carbachol or pilocarpine.



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  		Figure 1. Effect of pilocarpine on outflow facility in perfused human anterior segments. Data are the means of the ratios of Cd/Co ± SEM. n, number of eyes.

 


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  		Figure 2. Effect of aceclidine on outflow facility in perfused human anterior segments. Data are the means of the ratios of Cd/Co ± SEM. n, number of eyes.

 


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  		Figure 3. Effect of carbachol on outflow facility in perfused human anterior segments. Data are the means of the ratios of Cd/Co ± SEM. n, number of eyes.

 
Although each of the three agonists used in this study has been well established as a muscarinic agonist, we conducted blocking studies with atropine to ensure that the effects observed were due to stimulation of muscarinic receptors. For these studies we used 10-6 M atropine to block the outflow effects of 10-7 M carbachol. These studies showed that atropine completely blocked the outflow-increasing effects of carbachol (Fig. 4) .



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  		Figure 4. Administration of 10-6 M atropine (ATR) completely blocked the outflow effects of 10-7 M carbachol (CARB). Data are the means of the ratios of Cd/Co ± SEM, number of eyes = 6.

 
Additional studies were performed to evaluate the effect of perfusion with a maximally effective dose of aceclidine (10-6 M) for 1 hour, removal of the drug, and perfusion with DMEM for 1 hour (to reverse the outflow effect), followed by a high dose (10-4 M) of aceclidine for 1 hour. The results are shown in Figure 5 . The experiment clearly demonstrates that 10-6 M aceclidine effectively increases outflow facility in the same eye that does not respond to 10-4 M aceclidine (Fig. 5) .



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  		Figure 5. Effects of low- and high-dose aceclidine (ACE) on outflow facility in perfused human anterior segments. Shown is a single representative experiment demonstrating a 42% increase in outflow facility with 10-6 M aceclidine, which was reversed when drug was removed. A subsequent dose of 10-4 M produced no effect on outflow facility.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results of the present study show that the human outflow apparatus is capable of responding directly to muscarinic agonists with an increase in outflow facility without an intact ciliary muscle and that this increase can be blocked by atropine. This result is in direct contradiction to our current understanding that the therapeutic mechanism of action of pilocarpine and echothiophate iodide in the treatment of glaucoma is exclusively via contraction of the ciliary muscle.

Our understanding of muscarinic agonist–induced outflow facility effects stems from the elegant studies of Kaufman and Bárány, which demonstrated very convincingly that muscarinic agonist–induced outflow facility increases are mediated by the contraction of the ciliary muscle in the monkey eye in vivo.1 In those studies, the proximal attachment of the ciliary muscle to the scleral spur was severed so that when the ciliary muscle contracted, it was no longer connected to the outflow pathways via the scleral spur. When a ciliary muscle–disinserted eye was challenged with relatively high concentrations of pilocarpine (e.g., 10-4 to 10-2 M) there was no increase in outflow facility, which led to the conclusion that an intact ciliary muscle is necessary for the outflow facility–increasing effect of muscarinics in primate eyes.

The current studies were conducted in a similar "disinserted" system where the ciliary muscle was dissected away from the scleral spur in the course of preparation for organ culture.10 Similar to the findings of Kaufman and Bárány, high concentrations of muscarinic agonists did not increase outflow facility in this system. However, lower concentrations (10-9 to 10-6 M) resulted in increased outflow facility with all three agonists tested. Interestingly, Kiland et al.25 recently reported that low doses of pilocarpine do not increase outflow facility in the monkey eye in vivo. It would be interesting to know whether lower concentrations would affect outflow facility in the disinserted eyes.

In both our study using human eyes and the study of Kaufman and Bárány using monkey eyes, ciliary muscle cells were probably still present in the outflow pathways, because Tamm and colleagues26 have shown that not all fibers of the ciliary muscle insert at the spur. Apparently there are fibers that traverse the trabecular meshwork and insert as far forward as Schwalbe’s line in both human and monkey eyes. It is not known at this time whether or not these ciliary muscle–derived cells are the same ones that express muscarinic receptors and are responsible for the outflow facility–increasing effects observed in this study. Lepple–Wienhues et al.8 and Wiederholt and colleagues7 9 have shown that trabecular meshwork strips are contractile8 and that muscarinic agonists actually relax precontracted strips in the calf eye.7 9 It is not clear at this time how these tissue bath experiments relate to the in situ and in vivo systems in the primate eye.

It is interesting to note that there is a biphasic effect of muscarinic agonists on outflow facility. Given the presence of multiple muscarinic receptors in the human trabecular meshwork,4 it may be that the biphasic responsiveness to the nonselective agonists used in this study is due to a stimulation of multiple receptor subtypes and their related second-messenger systems, which could work in an antagonistic manner. It is known that the receptors coded for by the m1, m3, and m5 subtype genes are coupled via a pertussis–insensitive G protein to activation of phospholipase C. Stimulation of these subtypes also induces the release of arachidonic acid.27 Although the receptors coded for by the m2 and m4 genes are coupled to a pertussis–sensitive G protein, which activates potassium channels and inhibits adenylate cyclase–mediated cellular events,28 it has also been shown that m1 and m3 receptors can increase adenylate cyclase activity via a ß, {gamma} protein subunit.29

Alternatively, the biphasic effect might be explained by a regulatory system similar to that described by Yousufzai and coworkers.30 In their studies using bovine ciliary muscle, muscarinic stimulation resulted in the formation of prostaglandins such as PGE2 and PGD2, which increase cAMP, and stimulation of inositol phosphate pathway. Interestingly, they provided evidence that prostaglandins may negatively regulate the phosphoinositide system, which could explain the mechanism of the biphasic effect we observed.

In conclusion, our studies demonstrate that an intact ciliary muscle is not necessary for muscarinic agonist–induced increases in outflow facility to occur in human eyes. Further studies exploring the receptor subtypes involved in this effect and identification of the responsible second-messenger systems will elucidate further the mechanism of muscarinic agonist–induced outflow facility changes in human eyes.


   Footnotes
 
Supported in part by National Eye Institute Grant EYO 7321; The Massachusetts Lions Eye Research Fund; and Research to Prevent Blindness.

Submitted for publication November 16, 1999; revised January 7, 2000; accepted January 11, 2000.

Commercial relationships policy: N.

Corresponding author: Kristine A. Erickson, Department of Ophthalmology, Boston University School of Medicine, 715 Albany Street, L-914, Boston, MA 02118. kerick{at}bu.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Kaufman, PL, Bárány, EH (1976) Loss of acute pilocarpine effect on outflow facility following surgical disinsertion and retrodisplacement of the ciliary muscle from the scleral spur in the cynomolgus monkey Invest Ophthalmol 15,793-807[Abstract/Free Full Text]
  2. Grierson, I, Lee, W, Abraham, S. (1978) Effects of pilocarpine on the morphology of the human outflow apparatus Br J Ophthalmol 62,302-313[Abstract/Free Full Text]
  3. Shade, DL, Clark, AF, Pang, IH (1996) Effects of muscarinic agents on cultured human trabecular meshwork cells Exp Eye Res 62,201-210[Medline][Order article via Infotrieve]
  4. Gupta, N, Drance, SM, McAllister, R, Prasad, S, Rootman, J, Cynader, MS (1994) Localization of M3 muscarinic receptor subtype and mRNA in the human eye Ophthalmic Res 26,207-213[Medline][Order article via Infotrieve]
  5. Erickson, K. (1994) Muscarinic receptors in the ciliary muscle and outflow pathways Drance, SM eds. Drug Receptors in the Eye Williams & Wilkins Baltimore.
  6. deKater, A, Shahsafaei, A, Epstein, DL. (1992) Localization of smooth muscle and nonmuscle actin isoforms in the human aqueous outflow in the human aqueous outflow pathway Invest Ophthalmol Vis Sci. 33,424-429[Abstract/Free Full Text]
  7. Wiederholt, M, Lepple–Wienhues, A, Stahl, F. (1993) Contractile properties of trabecular meshwork and ciliary muscle Lütjen–Drecoll, E Rohen, JW eds. Basic Aspects of Glaucoma Research III ,287-306 Schattauer Stuttgart.
  8. Lepple–Wienhues, A, Stahl, F, Wiederholt, M. (1991) Differential smooth muscle-like contractile properties of trabecular meshwork and ciliary muscle Exp Eye Res 53,33-38[Medline][Order article via Infotrieve]
  9. Wiederholt, M, Schafer, R, Wagner, U, Lepple–Wienhues, A. (1996) Contractile response of the isolated trabecular meshwork and ciliary muscle to cholinergic and adrenergic agents Ger J Ophthalmol 5,146-153[Medline][Order article via Infotrieve]
  10. Erickson–Lamy, K, Rohen, JW, Grant, WM (1991) Outflow facility studies in the perfused human ocular anterior segment Exp Eye Res 52,723-731[Medline][Order article via Infotrieve]
  11. Erickson–Lamy, K, Nathanson, JA (1992) Epinephrine increases facility of outflow and cAMP content in the human eye in vitro Invest Ophthalmol Vis Sci 33,2672-2678[Abstract/Free Full Text]
  12. Erickson–Lamy, K, Schroeder, A. (1990) Dissociation between the effect of aceclidine on outflow facility and accommodation Exp Eye Res 50,143-147[Medline][Order article via Infotrieve]
  13. Erickson, KA, Schroeder, A, Netland, PA (1995) Verapamil increases outflow facility in the human eye Exp Eye Res 61,565-567[Medline][Order article via Infotrieve]
  14. Erickson, K, Liang, L, Shum, P, Nathanson, JA (1994) Adrenergic regulation of aqueous outflow J Ocul Pharmacol 10,241-252[Medline][Order article via Infotrieve]
  15. Liang, LL, Epstein, DL, de Kater, AW, Shahsafaei, A, Erickson–Lamy, KA (1992) Ethacrynic acid increases facility of outflow in the human eye in vitro Arch Ophthalmol 110,106-109[Abstract/Free Full Text]
  16. Erickson, KA, Nathanson, JA (1992) Epinephrine increases facility of outflow and cyclic AMP content in the human eye in vitro Invest Ophthalmol Vis Sci 33,2672-2678
  17. Erickson–Lamy, KA, Nathanson, JA (1993) The effects of endothelin, atrial natriuretic factor and nitroglycerin on outflow facility in the primate eye Lütjen–Drecoll, E eds. Basic Aspects of Glaucoma Research II ,275-285 Shattauer–Verlag Mainz, Germany.
  18. Schuman, JS, Erickson, K, Nathanson, JA (1994) Nitrovasodilator effects on intraocular pressure and outflow facility in monkeys Exp Eye Res 58,99-105[Medline][Order article via Infotrieve]
  19. Erickson–Lamy, KA, Rohen, JW, Grant, WM (1988) Outflow facility studies in the perfused bovine aqueous outflow pathways Curr Eye Res 7,799-807[Medline][Order article via Infotrieve]
  20. Kaufman, PL, Bàràny, EH (1977) Cytochalasin B reversibly increases outflow facility in the eye of the cynomolgus monkey Invest Ophthalmol Vis Sci 16,47-53[Abstract/Free Full Text]
  21. Kaufman, PL, Bill, A, Bàràny, EH (1977) Effect of cytochalasin B on con-ventional drainage of aqueous humor in the cynomolgus monkey Exp Eye Res 25(suppl),411-414
  22. Kaufman, PL, Erickson, KA (1982) Cytochalasin B and D dose-outflow facility response relationships in the cynomolgus monkey Invest Ophthalmol Vis Sci 23,646-650[Abstract/Free Full Text]
  23. Erickson–Lamy, K, Schroeder, AM, Bassett–Chu, S, Epstein, DL (1990) Absence of time-dependent facility increase ("washout") in the perfused enucleated human eye Invest Ophthalmol Vis Sci 31,2384-2388[Abstract/Free Full Text]
  24. Al–Aswad, LA, Gong, H, Lee, D, et al (1999) Effects of Na-K-2Cl cotransport regulators on outflow facility in calf and human eyes in vitro Invest Ophthalmol Vis Sci 40,1695-1701[Abstract/Free Full Text]
  25. Kiland, JA, Hubbard, WC, Kaufman, PL. (1999) Low doses of pilocarpine do not significantly increase outflow facility in the cynomolgus monkey [ARVO Abstract] Invest Ophthalmol Vis Sci 40(4),S171Abstract nr 915
  26. Tamm, E, Flügel, C, Stefani, FH, Rohen, JW (1992) Contractile cells in human scleral spur Exp Eye Res 54,531-543[Medline][Order article via Infotrieve]
  27. Lechleiter, J, Peralta, E, Clapham, D. (1989) Diverse functions of muscarinic acetylcholine receptor subtypes Trends Pharm Sci 34(suppl),8
  28. Berrie, C, Hawkins, PT, Stephens, LR, Harden, TK (1989) Phosphatidylinositol 4,5-bisphosphate hydrolysis in turkey erythrocytes is regulated by P2y purinoceptors Mol Pharmacol 35,526-532[Abstract]
  29. Baumgold, J, Paek, R, Yasumoto, T. (1992) Agents that stimulate phosphoinositide turnover also elevate cAMP in SK-N-SH human neuroblastoma cells Life Sci 50,1755-1759[Medline][Order article via Infotrieve]
  30. Yousufzai, S, Tachado, S, Abdel–Latif, A. (1990) Species differences in the effects of leukotriene D4 on inositol trisphosphate accumulation, cyclic AMP formation and contraction in iris sphincter of the mammalian eye Prostaglandins 39,227-240[Medline][Order article via Infotrieve]



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