Knockout of A₃ Adenosine Receptors Reduces Mouse Intraocular Pressure

Abstract

purpose. To test the putative role of A₃ adenosine receptors (ARs) in modulating intraocular pressure (IOP).

methods. IOP was monitored for up to 32 minutes in A₃-knockout (A₃AR^−/−) and A₃AR^+/+ control mice by the servo-null approach. The IOP responses to adenosine, A₃AR agonists and A₃AR antagonists were studied singly or in combination in both strains.

results. IOP was significantly lower in A₃AR^−/− mice (12.9 ± 0.7 mm Hg) than in A₃AR^+/+ control animals (17.4 ± 0.6 mm Hg). The nonselective AR agonist adenosine produced a much smaller increase in IOP (2.2 ± 0.8 mm Hg) in the knockout than in A₃AR^+/+ control mice (14.9 ± 2.4 mm Hg). The A₃-selective agonist IB-MECA did not affect IOP in A₃-knockout mice, but raised it in A₃AR^+/+ mice. The highly selective A₃AR antagonist MRS 1191 did not affect IOP in A₃AR^−/− mice, but lowered it in A₃AR^+/+ control mice. Preadministering MRS 1191 did not affect the small adenosine-triggered increase in IOP in A₃AR^−/− mice, but markedly attenuated adenosine’s effects on IOP in A₃AR^+/+ control mice. MRS 1523, an A₃AR antagonist less selective than MRS 1191 in rats, decreased IOP in both A₃AR^−/− and A₃AR^+/+ animals. As in black Swiss outbred mice and other mammalian species, reducing aqueous humor inflow with acetazolamide lowered IOP and administering water intraperitoneally increased IOP in both A₃AR^−/− and A₃AR^+/+ mice.

conclusions. The reduced IOP and altered purinergic responses of IOP in A₃AR knockout mice support the conclusion that A₃ARs contribute to the regulation of IOP.

The mouse, a suitable nonprimate mammal for studying genetic control of physiologic and pharmacologic function, ¹ is particularly favorable for investigating the pharmacology of aqueous humor dynamics because of the structural parallels of the aqueous humor outflow pathways ² and the similar functional responses to drugs that inhibit aqueous humor inflow and facilitate outflow ³ in the human. The mouse model is also the most accessible mammalian system for studying the relationship of gene function to phenotypic expression of glaucoma. Advances in understanding aqueous humor dynamics would facilitate developing novel drugs for treating glaucoma, because lowering intraocular pressure (IOP) is the only intervention documented to reduce the progression of blindness associated with this disease. ^{4 5 6}  

Progress in studying the mouse has long been impeded by the difficulty in measuring IOP in this species because its anterior chamber volume is only 2 to 4 μL. ^{3 7} The adaptation of the servo-null micropipette system (SNMS) has overcome the challenge of studying this small eye, permitting reliable monitoring of mouse IOP over periods as long as 45 minutes. ³ With this technique, we have measured IOP responses to subtype-specific adenosine-receptor (AR) agonists and antagonists. ⁸ Drugs activating A₁ and A₂ subtype adenosine receptors have been reported to lower and increase IOP, respectively, in rabbits ^{9 10} and monkeys, ¹¹ an effect ascribed in monkeys entirely to actions on aqueous humor outflow. ¹¹ In rabbits, the initial decrease in IOP has been reported to be mediated by a transient reduction in aqueous humor inflow, but the later ocular hypotensive effect appears mediated by facilitating outflow. ¹² In contrast, agonists of A₃ subtype adenosine receptors activate Cl⁻ channels of the nonpigmented ciliary epithelial cells, ^{13 14} a critical step in aqueous humor secretion in vivo and an action predicted to enhance inflow and consequently to increase IOP. Consistent with this hypothesis, agonists and antagonists of A₃ subtype ARs indeed increase and decrease IOP, respectively, in the mouse. ⁸ Further, the large increase in mouse IOP triggered by applying adenosine is largely prevented by preapplication of A₃AR antagonists. These findings suggest a central role for A₃ARs in IOP regulation. In view of potential cross-reactivity of drugs with other adenosine receptor subtypes, we have now further tested the putative role of A₃ARs by studying effects on IOP of purinergic drugs in A₃AR-knockout mice. 

Materials and Methods

Animals

Mixed-sex A₃-knockout (A₃AR^−/− congenic on C57Bl/6 [N12] ^{15 16} ) mice were obtained from The Merck Research Laboratories (West Point, PA) through Taconic, Inc. (Germantown, NY). The A₃AR^−/− mice appear phenotypically normal and their nonocular responses have been studied by several other investigators. ^{15 16 17 18 19} Control C57Bl/6 A₃AR^+/+ animals were purchased from Taconic. The mice, 7 to 9 weeks of age, were maintained under 12-hour light–dark illumination cycle with light onset at 7 AM, and were provided unrestricted access to food and water. All procedures conformed to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. 

Anesthesia

Mice were anesthetized with intraperitoneal ketamine (250 mg/kg) supplemented by topical proparacaine HCl 0.5% (Allergan, Hormigueros, Puerto Rico) for the IOP measurements, which were conducted between 1 PM and 6 PM. 

Measurement of IOP

IOP was monitored with the SNMS. ³ As described in detail and validated previously, ³ the SNMS incorporates an exploring 5-μm tip-diameter micropipette filled with 3 M KCl solution that ensures that the resistance of the fluid within the tip is much lower than that of the extracellular fluid. After advance of the tip across the cornea, the increased hydrostatic pressure in the anterior chamber forces aqueous humor into the micropipette. The consequent increase in micropipette resistance activates a vacuum pressure pump that generates counterpressure to restore the position of the aqueous humor/KCl interface and thus the initial electrical resistance. This counterpressure is equal to the increase in hydrostatic pressure encountered by the micropipette tip as it enters aqueous humor (i.e., the IOP). 

Mean IOP was calculated in each mouse by averaging 3 to 5 minutes of recorded data (acquired at 3 Hz) before and after drug application. Thus, each mean was obtained from 540 to 900 measurements. Based on the rapidity with which topical drugs affect IOP in mice, ³ data obtained after drug treatment were included for analysis beginning approximately 5 to 8 minutes after topical drug application. As the response to intraperitoneal acetazolamide was slower than that to the topical agents, data reduction was initiated approximately 15 minutes after applying this drug. 

Drugs

Drugs were applied topically in 10-μL droplets with a pipette (Eppendorf, Fremont, CA) at the stated concentrations. ⁸ Concentrations and corresponding doses are summarized in Tables 1 and 2 . Stock solutions were prepared in dimethyl sulfoxide (DMSO). The final droplet solution was isotonic saline (310 mOsm) containing 1% to 2% DMSO and 0.003% benzalkonium chloride (Sigma, St. Louis, MO) to enhance topical drug penetration. ²⁰ The vehicle solution containing DMSO (2%–8%)-benzalkonium (0.03%) itself has no effect on mouse IOP. ^{3 8} Based on prior studies, drugs applied topically by this method probably alter IOP by local ocular, not systemic, actions. ^{3 8 21}  

Adenosine, IB-MECA, MRS 1191, and MRS 1523 were obtained from RBI Sigma (St. Louis, MO), and the carbonic anhydrase inhibitor acetazolamide was purchased from Bedford Laboratories (Bedford, OH). 

Data Analysis

Unless otherwise stated, results are reported as the mean ± SEM. Both baseline and drug-induced changes in IOP were found to be normally distributed by the Kolmogorov-Smirnov test in each of the current series of experiments. The probability of the null hypothesis was estimated with the paired, two-tailed Student’s t-test for comparing drug response with baseline. 

To maximize the information obtained with the transgenic mice, both eyes were usually studied (44 eyes from 24 mice) during the same anesthetic period within an interval of 0.5 hour or more, commonly in sessions separated by at least 4 days. Both eyes were also studied with control A₃AR^+/+ mice (60 eyes from 43 animals). Each eye was studied only once. On the basis of direct comparisons of mouse IOP in right and left eyes, ³ the intereye correlation coefficient is estimated to be 0.86. To use the most conservative approach to account for intereye correlations, we assumed all data to have been obtained from both eyes of individual mice and that the physiological and pharmacologic responses of the two eyes would show the same high intereye correlation in individual mice. With this approach, the t-statistic is divided by (1.86)^1/2 to estimate the least upper limit to the probability (P) of the null hypothesis. ²² We followed common practice in defining significance at the 0.05 probability level, but have provided the estimates of P for all experimental series of measurements. 

Results

Baseline Levels

Baseline IOP in the A₃AR-knockout mice was 12.9 ± 0.7 mm Hg (n = 44 eyes). This was lower than the mean IOP in A₃AR^+/+ C57Bl/6 mice (17.4 ± 0.6 mm Hg, n = 60, P < 0.001). For purposes of comparison, the IOP in the A₃AR^+/+ control mice was similar to the mean (16.0 ± 0.3 mm Hg) for 292 black Swiss outbred mice we examined in prior studies ^{3 8 21} (Fig. 1) . 

A₃AR Agonists

The nonselective agonist adenosine increased IOP in the knockout mice by 2.2 ± 0.8 mm Hg at an applied droplet concentration of 100 μM and by 5.8 ± 1.8 mm Hg at a concentration of 2 mM (Table 1 , Figs. 2A 3 ). In contrast, 100 μM adenosine increased IOP in the A₃AR^+/+ C57Bl/6 mice by 14.9 ± 2.4 mm Hg (Table 2 ; Figs. 2B 3 ). We have reported that topical application of adenosine at 100-μM and 2-mM concentrations increases IOP in black Swiss outbred mice by 21.2 ± 3.2 and 16.4 ± 2.9 mm Hg, respectively. ⁸  

Topical application of the selective A₃AR agonist IB-MECA did not affect IOP in the transgenic mice at a droplet concentration of 140 nM (Table 1 , Figs. 3 4 ). In contrast, the same concentration of IB-MECA increased IOP by 8.3 ± 1.3 mm Hg in A₃AR^+/+ C57Bl/6 mice (Table 2 , Fig. 3 ). 

A₃AR Antagonists

The highly selective antagonist of both human and murine A₃ARs MRS 1191 ^{23 24} (25 μM) had no effect on baseline IOP in the A₃AR^−/− mice (Table 1 , Figs. 2C 3 ). In contrast, MRS 1191 at the same concentration reduced IOP in the A₃AR^+/+ C57Bl/6 mice by 7.0 ± 0.9 mm Hg (Table 2 , Figs. 2D 3 ); previously, it had reduced IOP in black Swiss outbred mice by 6.3 ± 0.7 mm Hg. ⁸  

After pretreatment with 25 μM MRS 1191, 100 μM adenosine increased IOP by 2.5 ± 0.9 mm Hg in the A₃AR^−/− mice (Table 1 , Fig. 2C ), an increase identical with that in mice without pretreatment (P > 0.8). In contrast, the same concentration of MRS 1191 nearly abolished the subsequent response of the A₃AR^+/+ mice to 100 μM adenosine (Table 2 , Fig. 2D ) and was previously found to reduce the adenosine response in black Swiss outbred mice markedly. ⁸  

The A₃AR antagonist MRS 1523, ²⁵ structurally dissimilar to MRS 1191, is as effective as MRS 1191 in lowering IOP in black Swiss outbred mice. ⁸ MRS 1523 (400 μM) reduced baseline IOP in the A₃AR^−/− mice by 3.6 ± 0.6 mm Hg (Table 1 , Fig. 3 ) but reduced the IOP in the A₃AR^+/+ mice to a significantly greater extent (9.8 ± 1.1 mm Hg; P < 0.005; Table 2 , Fig. 3 ). Pretreatment with MRS 1523 did not alter the magnitude of the subsequent response to 100 μM adenosine in the knockout mice (Table 1) . 

Nonpurinergic Modifications

The low basal IOP levels in A₃AR^−/− mice, particularly in the context of the nonlinear IOP-volume relationships observed in larger mammals, ²⁶ may attenuate IOP responses of the knockout mice to all perturbations. We assessed IOP responsiveness of A₃AR^−/− mice by measuring the responses both to reducing and increasing fluid flow into the eye. First, the carbonic anhydrase inhibitor acetazolamide, which reduces aqueous humor secretion into mammalian eyes, lowered IOP in the A₃AR^+/+ mice (by 6.0 ± 0.8 mm Hg) at an intraperitoneal dose of 8.3 mg/kg (Table 2) . At the same dose, acetazolamide also reduced IOP in the A₃AR^−/− mice (by 2.2 ± 0.4 mm Hg, Table 1 , Fig. 5 ). Second, increasing water flow into the eye by intraperitoneal injection of a water load produced a large increase in IOP (Figs. 2A 4 5) , conforming to the general ocular response to a water load that also occurs in wild-type mice ³ and in A₃AR^+/+ C57Bl/6 mice (Fig. 2D) . 

Discussion

A₃AR mRNA is expressed in cultured human nonpigmented epithelial (NPE) cells and rabbit ciliary processes. ¹⁴ Functional studies, both electrophysiologic and volumetric, of cultured human and native bovine cells and rabbit iris-ciliary body have established that A₃ARs regulate Cl⁻ channel activity in the NPE ciliary cells, the innermost layer of the epithelial bilayer that secretes aqueous humor. Both adenosine and A₃AR-selective agonists activate Cl⁻ channels in NPE cells, ^{13 14 27} and A₃AR-selective antagonists block adenosine-triggered Cl⁻ channel activation. ^{14 27} A₃AR regulation of Cl⁻ channels is not limited to NPE cells. ²⁸ Although A₃ agonists are known to reduce cAMP levels, increase free intracellular Ca²⁺ concentration and alter PKC activity, the transduction mechanism linking agonist occupancy of A₃ARs and Cl⁻ channel activation is unidentified. ^{13 14 27} The observations that A₃AR agonists activate Cl⁻ channels led to the hypothesis that these agonists would increase aqueous humor secretion and thereby IOP in vivo, and that A₃AR antagonists would exert the opposite effects. These predicted effects on IOP were indeed observed in black Swiss outbred mice, ⁸ by using the SNMS. The SNMS has been validated for monitoring mouse IOP by comparing measured and imposed pressures in the same eye, by successive measurements in the same eye, by comparing IOP in right and left eyes, and by measuring the responses to application of anisosmotic solutions and drugs that alter IOP in other species through well-established effects on aqueous humor inflow and outflow. ³  

The present results with A₃AR^−/− mice confirm a role for A₃ARs in regulating aqueous humor dynamics. First, A₃AR^−/− mice displayed a lower baseline IOP than either A₃AR^+/+ control or black Swiss outbred mice, suggesting a role for A₃ARs in maintaining basal IOP levels. Second, the selective A₃AR antagonist MRS 1191 had no effect on IOP in A₃AR^−/− mice, but reduced IOP in A₃AR^+/+ control mice to the same extent as in black Swiss outbred mice. ⁸ Third, the physiologic and nonselective agonist adenosine produced a modest elevation of IOP in the A₃AR^−/− (2.2 ± 0.8 mm Hg) that was 7 times lower than in A₃AR^+/+ control mice (14.9 ± 2.4 mm Hg) and 10 times lower than in black Swiss outbred mice (21.2 ± 3.2 mm Hg) at a topical droplet concentration of 100 μM. ⁸ Fourth, the small adenosine-activated increase in IOP in A₃AR^−/− mice was not affected by prior application of the selective A₃AR antagonist MRS 1191, in contrast to its reduction of the adenosine effect in both the A₃AR^+/+ control animals and black Swiss outbred mice. ⁸ Fifth, the selective A₃AR agonist IB-MECA exerted no effect on IOP in the A₃AR^−/− mice, in contrast to the elevation of IOP noted in both the A₃AR^+/+ control mice (Table 2 , Fig. 3 ) and in black Swiss outbred mice. ⁸  

In contrast to the A₃AR antagonist MRS 1191, MRS 1523 lowered IOP in A₃AR^−/− mice, but its effect in knockout mice was only approximately one third of its ocular hypotensive effect in A₃AR^+/+ control animals. Although its selectivity for mouse receptors is not known, the relative selectivity of MRS 1523 for A₃ARs over A_2AARs in the rat has been estimated to lie between 4-fold ²² and 16-fold. ²⁹ MRS 1191 is presently the most selective A₃AR antagonist both in humans and in the rat (28-fold in binding ²⁴ ); it blocks A₃AR responses in mouse cells—specifically, in PGT-β mouse pineal gland tumor cells. ³⁰ The reduced selectivity of MRS 1523 raises the possibility that this antagonist may have cross-occupied A_2AARs, which would also have reduced IOP. ^{8 9 10 11} An alternative interpretation is that either the slight ocular hypertensive effect of adenosine or the ocular hypotensive effect of MRS 1523 in the A₃AR^−/− mice was mediated by a mechanism as yet unidentified. 

As discussed elsewhere, ^{8 21} the very small volume of the mouse anterior chamber (∼2–4 μL ^{3 7} ) precludes measurement of drug concentrations in aqueous humor after topical administration. We have, however, compared minimally effective droplet concentrations of purinergic drugs with published functional estimates of their binding to compute a penetrance (the aqueous-to-droplet concentration ratio). That penetrance is commonly 1:100–1000. ⁸ Using this index, application of adenosine at a droplet concentration of 100 μM (Tables 1 2) may have corresponded to 0.1 to 1.0 μM in the aqueous humor. Over this concentration range, adenosine is likely to occupy A₁-, A_2A-, and A₃ARs, but not A_2BARs. ³⁰  

In summary, the reduction in baseline IOP, the complete absence of an ocular hypertensive response to the selective A₃AR agonist IB-MECA, the complete absence of an ocular hypotensive response to the selective A₃AR antagonist MRS 1191, and the highly attenuated ocular hypertensive response to adenosine all support the conclusion that adenosine acts through A₃ARs, at least in part, to regulate IOP, and that A₃AR antagonists may be useful in treating glaucoma. In addition, these results further support the utility of the SNMS for studying IOP in mice. 

 Supported in part by Research Grants EY08343 (MMC) and EY013624 (MMC) and Core Grant EY01583 from the National Eye Institute, and The Paul and Evanina Mackall Foundation Trust (RAS). RAS is a recipient of a Research to Prevent Blindness Physician-Scientist Merit Award.

 Submitted for publication March 4, 2002; revised May 20, 2002; accepted May 30, 2002.

 Commercial relationships policy: P (RAS, MMC); N (MYA).

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Effects of Drugs on IOP in A₃AR^−/− Mice

Drug	Class	n	Concentration (dose)	ΔIOP Versus Baseline	ΔIOP Versus Preceding Drug	P
Adenosine	Nonspecific P1 agonist	6	100 μM (316 ng)	+2.2 ± 0.8		>0.05
		6	2 mM (6.32 μg)		+5.8 ± 1.8	<0.05
IB-MECA	A3 agonist	6	140 nM (714 pg)	+0.1 ± 0.3		>0.95
MRS 1191	A3 antagonist	9	25 μM (94 ng)	−0.3 ± 1.4		>0.8
Adenosine after MRS1191		9	100 μM (316 ng)		+2.5 ± 0.9	<0.02
MRS 1523	A2A-, A3-antagonist	7	400 μM (1.60 μg)	−3.6 ± 0.6		<0.005
Adenosine after MRS1523		6	100 μM (316 ng)		+2.5 ± 0.9	>0.05
Acetazolamide	Carbonic anhydrase inhibitor	6	8.3 mg/kg*	−2.2 ± 0.4		<0.005

*  Acetazolamide was injected intraperitoneally.

Table 2.

 

Effects of Drugs on IOP in A₃AR^+/+ Mice

Drug	Class	n	Concentration (dose)	ΔIOP Versus Baseline	ΔIOP Versus Preceding Drug	P
Adenosine	Nonspecific P1 agonist	8	100 μM (316 ng)	+14.9 ± 2.4		<0.005
IB-MECA	A3 agonist	7	140 nM (714 pg)	+8.3 ± 1.3		<0.005
MRS 1191	A3 antagonist	24	25 μM (94 ng)	−7.0 ± 0.9		<0.001
Adenosine after MRS 1191		8	100 μM (316 ng)		+4.5 ± 2.2	>0.1
MRS 1523	A2A-, A3-antagonist	7	400 μM (1.60 μg)	−9.8 ± 1.1		<0.005
Acetazolamide	Carbonic anhydrase inhibitor	5	8.3 mg/kg*	−6.0 ± 0.8		<0.01

*  Acetazolamide was injected intraperitoneally.

Figure 1.

 

Baseline IOP in A₃AR^−/− (n = 44) and A₃AR^+/+ control (n = 42) mice in the present series and in black Swiss outbred mice (n = 292) measured in previous studies. ^{3 8 21} Central horizontal lines: medians; lower and upper lines: all data between the 25th and 75th percentiles; whiskers: data range between the 10th and 90th percentiles. Circles are individual data points lying outside this range. The IOP in the A₃AR^−/− mice was significantly lower than that in the two control groups.

Figure 2.

 

Effects of the nonselective AR agonist adenosine and the A₃-selective antagonist MRS 1191 on IOP in A₃AR^−/− and A₃AR^+/+ mice. Each trace presented in Figures 2 4 and 5 was obtained from continuous measurement of a single mouse. (A) Adenosine had little effect on IOP in A₃AR^−/− mice at a droplet concentration of 100 μM or 2 mM, whereas intraperitoneal water elevated IOP, as noted in wild-type mice. (B) In contrast, the lower adenosine concentration markedly elevated IOP in control A₃AR^+/+ mice. (C) Application of 25 μM MRS 1191 did not alter baseline IOP in A₃AR^−/− mice and did not inhibit the subsequent slight response to 100 μM adenosine. (D) The same droplet concentration of MRS 1191 markedly lowered baseline IOP in control A₃AR^+/+ mice and strongly inhibited the subsequent response to 100 μM adenosine. Once again, intraperitoneal water produced the expected increase in IOP.

Figure 3.

 

Summary of changes in IOP produced by A₃AR agonists (adenosine and 1B-MECA) and A₃AR antagonists (MRS 1191 and MRS 1523) in A₃AR^−/− and A₃AR^+/+ mice. Further details are presented in Tables 1 and 2 .

Figure 4.

 

Effects of IB-MECA and intraperitoneal water on the IOP in an A₃AR^−/− mouse. Topical application of 140 riM IB-MECA had no effect, but intraperitoneal water triggered a characteristic increase in IOP.

Figure 5.

 

Effects of acetazolamide and intraperitoneal water on the IOP in an A₃AR^−/− mouse. Intraperitoneal acetazolamide lowered IOP, and subsequent intraperitoneal water elicited the expected increase in IOP.

The authors thank Marlene A. Jacobson and Merck Research Laboratories, West Point, PA, for providing the transgenic mice indispensable for this study; Maureen G. Maguire for help with the statistical analysis; and Kenneth A. Jacobson, Marlene A. Jacobson, Bruce L. Liang, and Jody R. Piltz-Seymour for helpful discussions.