Intraocular Pressure Responses to the Adenosine Agonist Cyclohexyladenosine: Evidence for a Dual Mechanism of Action

Abstract

purpose. Previous studies have shown that adenosine agonists are effective in reducing intraocular pressure (IOP). However, the mechanism(s) responsible for this ocular hypotensive effect has not been established. This study evaluates the relative contribution of changes in aqueous flow and outflow facility associated with the ocular hypotensive response to the adenosine agonist cyclohexyladenosine (CHA).

methods. New Zealand White rabbits were treated topically in one eye with the adenosine A₁ agonist CHA. Changes in IOP, aqueous flow, and total outflow facility at various times after CHA administration were then determined.

results. These studies demonstrated that CHA produces a dose-related reduction in IOP. Analysis of the dose–response curve revealed an ED₅₀ and a Hill coefficient of 87 μg and 1.9, respectively. Aqueous flow measurements demonstrated that 1.5 hours after CHA administration, aqueous flow was reduced by 35%. However, by 3.5 hours postdrug, no significant change in aqueous flow was observed. Measurement of the outflow facility found no significant change in facility 1.5 hours after CHA administration. However, by 3.5 hours after CHA administration, outflow facility was significantly increased by 85%.

conclusions. These data demonstrate that the adenosine agonist CHA lowers IOP in a dose-related fashion. This hypotensive action results from an early reduction in aqueous flow followed by a subsequent increase in outflow facility. This dual mechanism of action is consistent with analysis of CHA dose–response curve, which indicates that the reduction in IOP induced this agonist’s results from multiple mechanisms of action.

Biochemical, pharmacological, and molecular studies have demonstrated that four distinct populations of adenosine receptors exist: A₁, A_2a, A_2b, and A₃. ^{1 2 3 4} The activation of these receptors has been shown to modulate a number of diverse physiological functions. These include membrane transport, neurotransmission, blood flow, gene expression, and cellular differentiation and division. ^{5 6 7 8 9 10 11} In the anterior segment of the eye, molecular and pharmacological studies have confirmed the presence of A₁, A_2a, and A_2b receptors. ^{12 13 14 15} The administration of adenosine A₁ agonists has been shown to lower intraocular pressure (IOP) in rabbits and monkeys. ^{13 16} More recently, studies have shown that elevations in endogenous levels of adenosine in the aqueous humor lead to activation of adenosine receptors and reduction in IOP. ¹⁷ Therefore, adenosine A₁ receptors may play a physiological as well as a pharmacological role in the regulation of IOP. 

The mechanisms responsible for this adenosine receptor–mediated reduction in IOP have not been determined. Initial studies provided evidence that the reduction of pressure was associated with the decrease in aqueous flow. ¹³ However, other studies have shown that aqueous outflow is increased in animals after the administration of adenosine agonists. ^{16 18} The current studies were designed to evaluate if change in aqueous flow, outflow facility, or both are involved in the ocular hypotensive response to adenosine A₁ agonist cyclohexyladenosine (CHA). Results from these studies provide evidence that CHA-induced reduction in IOP result from an early reduction in aqueous flow, whereas the maintenance of this ocular hypotensive response results from an increase in outflow facility. 

Materials and Methods

Animals

The experimental animals used in this study were New Zealand White rabbits (2–3 kg), housed under standard laboratory conditions, with a 12-hour light and dark cycle. All animals had free access to food and water and were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

IOP Measurements

IOPs were measured in a masked fashion using a calibrated DigiLab Modular One pneumotonometer (Cambridge, MA). To minimize discomfort to the animal during tonometry, corneas were lightly anesthetized by the application of 10 μl of 0.1% proparacaine. After two baseline measurements (−0. 5 and 0 hours), the adenosine A₁ agonist cyclohexyladenosine CHA (Research Biochemical Inc., Natick, MA), or vehicle (20% DMSO) were all applied topically (50 μl) to one eye. IOPs were then measured at 0.5, 1, 2, 3, 4, and 5 hours postdrug. 

Aqueous Flow Measurements

Fluorotron master (OcuMetrics, Mountain View, CA) was used to measure aqueous fluorescein concentrations. The fluorometer was calibrated using a graded series of fluorescein solutions (10⁻⁹ to 10⁻⁶ g/ml). Fourteen hours before the start of fluorometry, 10 μl of a 2% fluorescein solution was applied to the cornea every 5 minutes for 60 minutes. On the day of the experiment, CHA (500 μg) or vehicle was applied unilaterally, and fluorescein concentrations were measured each half-hour from 1 to 2 and 3 to 4 hours after administration. Fluorometric measurements of anterior chamber and corneal fluorescein concentrations, along with estimates of anterior chamber and corneal volume were used to determine the rate of aqueous flow as previously described. The rate of aqueous flow was determined by the following equation: F = (ΔM/ΔT)/C _a, where ΔM is the change in total mass of fluorescein,Δ T is the time interval between measurements, and C _a is the average concentration of fluorescein in the anterior chamber during the time interval. 

Total Outflow Facility Measurement

Total outflow facility was determined by two-level constant pressure perfusion of the anterior chamber (3 and 13 mm Hg above spontaneous IOP) with Barany’s mock aqueous humor (NaCl 8 g/l, KCl 0.35 g/l, CaCl 0.17 g/l, MgCl₂ 64 mg/l, Na₂PO₄ 69 mg/l, NaH₂PO₄ 13.7 mg/l, glucose 1g/l). Rabbits were treated topically with CHA (500 μg) or vehicle. One or three hours after the administration of CHA, rabbits were then anesthetized with 33 mg/kg of ketamine and 6 mg/kg of Rompum, corneas were anesthetized by the application of 50 μl of 0.5% proparacaine, and the anterior chamber was cannulated with a single 26-gauge needle connected to perfusion apparatus. Outflow facility was then measured for 30 minutes starting at 1.25 or 3.25 hours postdrug. During this period four to five facility measurements were obtained and averaged to give the final value for each animal. All facility measurements were corrected for internal resistance of the perfusion apparatus. 

Data Analysis

Values are presented as mean ± SE. Drug-treated ipsilateral and contralateral responses were compared with corresponding ipsilateral and contralateral responses in vehicle-treated animals by means of Student’s t-test for nonpaired data. A P value of 0.05 was considered significant. Dose–response curves were analyzed by nonlinear regression analysis using Prism software (GraphPad Software Inc., San Diego, CA). The ED₅₀ and Hill coefficient were entered as variables in the dose–response equation and reported as best fit values. Starting values for the regression analysis were determined by visual inspection of the data. 

Results

The inset in Figure 1 shows the change in IOP in the ipsilateral eye after the administration of CHA (500 μg) or vehicle. Administration of CHA induced an initial hypertensive response of 2.2 ± 0.8 mm Hg at 0.5 hours. After this initial hypertension, a significant reduction in IOP of 4 to 8 mm Hg from 2 to 5 hours postdrug was measured. In the contralateral eye, a brief reduction in IOP of 2 to 3 mm Hg was observed 1 to 2 hours after CHA administration (data not shown). At doses lower than 500 μg of CHA no significant change in contralateral IOP was measured. No significant change in IOP from basal levels were observed in the ipsilateral or contralateral eye after vehicle (20% DMSO) administration. 

The dose–response relationships for the peak reduction in ipsilateral IOP for the adenosine A₁ agonist CHA is shown in Figure 1 . These data demonstrate that CHA produced a dose-related reduction in IOP. The ED₅₀ and response maximum for the dose–response curve were 87 ng and 8.1 mm Hg, respectivley. The Hill coefficient for the dose–response curve was 1.9. 

The effect of CHA (500 μg) or vehicle administration on aqueous flow is presented in Figure 2 . At 1.5 and 3.5 hours after vehicle administration, mean aqueous flows were 2.0 ± 0.27 and 2.1 ± 0.16 μl/min, respectively. At 1.5 hours post-CHA administration, aqueous flow was significantly reduced by 38% when compared with vehicle-treated animals. At 3.5 hours post-CHA administration, mean aqueous flow was reduced by 13% when compared with corresponding value from vehicle-treated animals; however, this reduction in flow was not significant. 

The effect of CHA (500 μg) or vehicle administration on outflow facility is presented in Figure 3 . At 1.5 and 3.5 hours after vehicle administration, mean outflow facility values were 0.22 ± 0.02 and 0.21 ± 0.01μ l/min/mm Hg, respectively. At 1.5 hours post-CHA administration, a small increase in outflow facility was measured; however, this change in facility was not significant. At 3.5 hours post-CHA administration, outflow facility had increased significantly by 85% when compared with vehicle-treated animals at identical times. 

Discussion

The present study confirms previous reports that topical administration of adenosine agonists produces a biphasic change in IOP: an initial hypertensive response followed by a prolonged reduction in IOP. Pharmacology studies have shown that the early hypertensive phase results from the activation of adenosine A_2b receptors, ¹⁴ whereas the ocular hypotensive response results from the activation of adenosine A₁ receptors. ¹³ However, studies designed to determine whether the reduction in IOP in these studies results from changes in aqueous flow or outflow facility have produced conflicting results. ^{13 16 18}  

The unilateral nature of the CHA-induced reductions in IOP indicates that the receptors responsible for the reduction in IOP are located in the anterior segment of the eye. Analysis of the CHA dose–response data demonstrated that the Hill coefficient for this response was 1.9. This deviation of the Hill coefficients from 1.0 indicates that the IOP response to CHA cannot be attributed to a single set of noninteracting receptors. Therefore, these data are consistent with the idea that at the time of the peak reduction in IOP, the adenosine A₁ agonists act at multiple sights within the anterior segment to lower IOP. Previous studies have shown that adenosine A₁ receptors are located on the ciliary epithelium, sympathetic fibers, and trabecular meshwork cells. ^{9 15 19}  

To understand how changes in aqueous flow and outflow facility contribute to adenosine A₁ receptor-mediated reduction in IOP, these parameters were measured at times corresponding to early (1.5 hours postdrug) and late (3.5 hours postdrug) phases of the ocular hypotensive response to CHA. Aqueous flow was significantly reduced during the early phase by 38%. However, by 3.5 hours postdrug no significant change in aqueous flow could be detected. As IOPs in these animals remained significantly below basal levels at 3.5 hours postdrug, the Goldmann equation predicts that outflow resistance or episeleral venous pressure must be reduced during this period. 

Analysis of outflow facility after CHA administration demonstrated that at 1.5 hours a trend toward small increases in outflow facility; however, this change was not significant. By 3.5 hours postdrug outflow, facility had increased significantly by 85% over vehicle-treated control animals. This increase in facility is similar to changes noted in previous studies and is sufficient to account for the reduction in IOP. ¹⁶  

In summary, these data demonstrate that adenosine agonists lower IOP in rabbits by a dual mechanism of action. This hypotensive action is composed of an early reduction in aqueous flow followed by a subsequent increase in outflow facility. This dual site of action is consistent with the Hill coefficients values calculated from dose–response curves being greater than one. This dual mechanism also makes the development of adenosine agonists attractive candidates for the treatment of ocular hypertension. 

 Supported by National Eye Institute Grant EY-09741 and Research to Prevent Blindness.

 Submitted for publication September 25, 2000; revised March 8, 2001; accepted March 19, 2001.

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Figure 1.

 

Dose–response curve for the peak reduction in IOP induced by adenosine A_l agonists CHA (n = 8–10). Solid line: best-fit of dose–response data by nonlinear regression analysis; inset: change in IOP induced by the topical administration of CHA (500 μg) or vehicle (20% DMSO) to New Zealand White rabbits. CHA or vehicle was administered unilaterally at t = 0. *Significant difference (P < 0.05) between the ipsilateral eye of treated rabbits compared with corresponding ipsilateral responses after vehicle administration (n = 10).

Figure 2.

 

Change in aqueous flow induced by CHA (500 μg) at 1.5 and 3.5 hours after agonist administration. *Significant difference (P < 0.05) between CHA-treated eyes and vehicle-treated eyes (20% DMSO) at identical times (n = 6).

Figure 3.

 

Change in outflow facility induced by CHA (500 μg) at 1.5 and 3.5 hours after agonist administration. *Significant difference (P < 0.05) between CHA-treated eyes and vehicle-treated eyes (20% DMSO) at identical times (n = 6).

The author thanks Melissa McAleer for her technical assistance in this study.