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

Abstract

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.

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 meshwork ³ and human trabecular meshwork tissue in situ ^{4 5} have muscarinic receptors. Moreover, meshwork cells with contractile properties have been demonstrated histologically ⁶ 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

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 previously ¹¹ 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 Canal ¹⁰ ), 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  

Results

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. 

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) . 

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) . 

Discussion

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 colleagues ²⁶ 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 colleagues ^{7 9} have shown that trabecular meshwork strips are contractile ⁸ 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 β, γ 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. 

 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.

 

Table 1.

 

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

M	Pilocarpine							Aceclidine							Carbachol
		n	Co ± SEM	Cd ± SEM	Cd/Co ± SEM	n	Co ± SEM	Cd ± SEM	Cd/Co ± SEM	n	Co ± SEM	Cd ± SEM	Cd/Co ± SEM
10−9	4	0.33 ± 0.04	0.40 ± 0.04	1.27 ± 0.12	6	0.54 ± 0.10	0.81 ± 0.19	1.47 ± 0.17*	6	0.31 ± 0.06	0.39 ± 0.06	1.34 ± 0.09, †
10−8	5	0.35 ± 0.04	0.44 ± 0.04	1.29 ± 0.03, §	5	0.47 ± 0.09	0.69 ± 0.16	1.68 ± 0.34	5	0.34 ± 0.06	0.43 ± 0.08	1.32 ± 0.15
10−7	5	0.35 ± 0.04	0.46 ± 0.09	1.30 ± 0.14	6	0.54 ± 0.10	1.00 ± 0.26	1.80 ± 0.20, †	6	0.31 ± 0.06	0.44 ± 0.08	1.50 ± 0.12, ‡
10−6	5	0.35 ± 0.04	0.47 ± 0.11	1.31 ± 0.18	6	0.54 ± 0.10	0.95 ± 0.19	1.98 ± 0.26, †	4	0.39 ± 0.05	0.51 ± 0.12	1.32 ± 0.19
10−4	6	0.20 ± 0.08	0.17 ± 0.06	0.95 ± 0.07	6	0.20 ± 0.06	0.22 ± 0.09	0.98 ± 0.12	5	0.23 ± 0.05	0.21 ± 0.03	1.02 ± 0.19
10−3	6	0.20 ± 0.08	0.17 ± 0.06	0.98 ± 0.14	6	0.20 ± 0.06	0.22 ± 0.10	0.96 ± 0.15	5	0.23 ± 0.05	0.18 ± 0.03	0.98 ± 0.29
10−2	6	0.20 ± 0.08	0.14 ± 0.03	0.89 ± 0.11	6	0.20 ± 0.06	0.22 ± 0.09	1.00 ± 0.16	5	0.23 ± 0.05	0.20 ± 0.04	0.98 ± 0.20
Control	9	0.27 ± 0.08	0.28 ± 0.08	1.02 ± 0.02

 Co indicates baseline outflow facility, and Cd indicates postdrug exchange outflow facility. Data are the means of the ratios of Cd/Co for n eyes; that is, for Cd/Co, the ratio of each eye’s pre- and postdrug facility was calculated first, and then the mean of individual ratios was determined. Thus, Cd/Co in the column above is a mean of ratios and not a ratio of means. As a result, Cd/Co is not the simple ratio of the preceding columns. The total number of eyes perfused with aceclidine, carbachol, and pilocarpine was 12, 11, and 11, respectively. Control indicates a comparison of outflow facility after 6 to 8 hours of perfusion baseline facility in eyes that did not receive any drug.

*   P < 0.05.

†   P < 0.02.

‡   P < 0.01.

§   P < 0.001.

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.

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.

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.

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.

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.