Effects of Ion Transport and Channel-Blocking Drugs on Aqueous Humor Formation in Isolated Bovine Eye

Abstract

purpose. To investigate the role of active chloride secretion by the ciliary epithelium in the formation of aqueous humor (AH), by using the in vitro perfused eye.

methods. Bovine eyes collected from an abattoir were cannulated through the ophthalmic artery and perfused with oxygenated Krebs’ solution at 37°C. Aqueous humor formation (AHF) was measured by the fluorescein-dilution technique. Drugs were added to the perfusate and/or to the anterior chamber.

results. NaK-adenosine triphosphatase (ATPase) inhibitor, ouabain (1.0 mM), produced a significant reduction in AHF by 46% and 42% when added to the stromal or aqueous side, respectively. When added to both sides (1.0 mM), it produced a reduction of 61%. Bumetanide (0.1 mM), a specific inhibitor of Na-K-2Cl cotransport, and furosemide (0.1 mM), a nonspecific anion transport inhibitor, produced 35% and 45% reductions when applied to the stromal side. DIDS (0.001–0.1 mM), which is believed to inhibit the Cl-HCO₃ exchanger, Na-HCO₃ cotransporter, and chloride channel, produced a dose-dependent reduction when added to the stromal side. The inhibition was 55% by the highest concentration used. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 0.1 mM), a chloride channel blocker in the nonpigmented cells, produced a 25% reduction when applied to the aqueous side. Acetazolamide (0.1 mM), a carbonic anhydrase inhibitor, applied to the stromal side, produced 31% reduction.

conclusions. At least 60% of the AH is formed by active secretion in bovine eyes. Transport of anions through the ciliary epithelium (CE), particularly the chloride ion, plays a crucial role in AHF.

Aqueous humor (AH) is formed by the double-layered epithelium of the ciliary body through a process that is generally assumed to require active metabolism. ^{1 2} This secretion is based on the movement of water and electrolytes from the ciliary stroma across the epithelial cell layer, into the posterior chamber. This layer consists of the outer pigmented epithelium (PE), with its basement membrane resting on the ciliary stroma, and the inner nonpigmented epithelium (NPE), with its basement membrane facing the posterior chamber. Thus, unusual in secretory epithelia, these cells lay apex to apex. ³ Water appears to move passively across this layer by osmosis, after ion movements that are thought to be ultimately driven by NaK-adenosine triphosphatase (ATPase). Among the other ions involved, there may be an important role for chloride, whose efflux has been proposed as the rate-limiting step in transepithelial chloride transport. ⁴ Net transepithelial flux of chloride ions has been reported in the ciliary body of rabbit, ^{5 6} cat, ⁷ toad, ⁸ and ox. ⁹  

Recent studies have identified a number of cotransporter exchangers, ion channels, and pumps in the ciliary epithelium (CE). These include the Na-K-2Cl (NKCC) cotransporter and the parallel Cl-HCO₃ and Na-H antiport, ¹⁰ which are indirectly coupled to intracellular pH and carbonic anhydrase (CA). Previously we used a dissected preparation of bovine ciliary body to study ion transport and its inhibition. The present study extends this work to a whole eye model. Our purpose was to investigate the effect of transport inhibitors on the rate of aqueous humor formation (AHF) in vitro and to correlate the AHF with ion transport activities in bovine tissue. 

Methods

Preparation of Isolated Eye

Bovine eyes obtained from an abattoir were transported to the laboratory on ice. The perfusion of the eyes and estimation of AHF rate were conducted according to slight modification of the methods described earlier. ^{11 12} Excess adnexal tissue was trimmed away from the eye, but care was taken not to damage blood vessels running over the posterior surface of the globe and along the optic nerve. A few millimeters of each extraocular muscle was left attached to the globe. The ophthalmic artery was cleared of fat and cannulated distal to the point at which it divides to form the two long posterior ciliary arteries. The eye was placed in a circulating warming jacket maintained at 37°C and covered with an insulated plastic cup, and the ophthalmic artery was perfused with Krebs’ solution at 37°C, containing (mM): NaCl, 118; KCl, 4.7; MgSO₄, 1.2; CaCl₂, 2.5; NaHCO₃, 25; KH₂PO₄, 1.2; glucose, 11.5; and ascorbate 0.05. The pH of this solution was adjusted to 7.4 by bubbling with O₂ containing 5% CO₂. 

The experiments in the present study were conducted with a digital peristaltic pump (505S; Watson-Marlow Bredel, Falmouth, UK) to induce flow through the vasculature, with arterial perfusion pressure recorded by a transducer (model 60-3003; Harvard Apparatus, South Natick, MA) and pen recorder (BD 112; Kipp & Zonen, Bohemia, NY). Flow was commenced at 0.2 mL/min and increased in 10 to 20 increments to 3 mL/min in the first hour. During this period, the perfusion pressure was checked continuously, and preparations were said to be valid if the pressure was at or below 140 mm Hg. 

Estimation of the Rate of AHF

After the perfusion was set up and the eye appeared firm, the anterior chamber was cannulated with three 23-gauge needles. The first needle was connected by silicon rubber tubing (inside diameter, 0.5 mm; wall thickness, 1.8 mm) through a peristaltic pump (Watson-Marlow Bredel) to the cuvette of a spectrophotometer (Spectronic 2000; Amersham Pharmacia Biotech, Amersham, UK), returning to the anterior chamber through a second 23-gauge needle. This constituted the anterior chamber circulating system. The third needle connected the anterior chamber to a water manometer to measure the intraocular pressure (IOP). The points of the two circulating needles in the chamber were kept wide apart to optimize mixing. 

All anterior tubing and the cuvette were prefilled with 1.04 mL of an AH substitute comprising (mM): NaCl, 110; KCl, 3; CaCl₂, 1.4; MgCl₂, 0.5; KH₂PO₄, 0.9; NaHCO₃, 30; glucose, 6; ascorbate, 3; and fluorescein sodium, 0.000625%. The pH of this solution was adjusted to 7.6 by bubbling with 95% O₂ and 5% CO₂. This fluid was circulated through the anterior chamber at a rate of 0.25 mL/min. The absorbance was recorded at 488 nm every 5 minutes, and steady state was achieved in 20 to 40 minutes. 

AHF rate was estimated from the rate of fluorescein dilution (decrease in absorbance) of the anterior chamber circulating system. When the rate of formation is constant, a plot of log_e [absorbance of fluorescein] against time (in minutes) is a straight line with a slope that gives the rate constant (K _out per minute) for AHF. Regression lines representing the dilution of fluorescein in the aqueous were constructed using an ANOVA statistical package (Minitab, State College, PA). The K _out for the initial 30 minutes—that is, before the addition of drugs—was used as the control value to compare against that in the subsequent (40 minutes to 1 hour) drug-induced conditions. Drugs intended to act on the blood or PE side were added to the perfusate with a pipette. Drugs intended to act on the aqueous or NPE side were injected into the lumen of the anterior chamber circulating tube, through a sealable rubber connector, with a microsyringe, in volumes of 3 to 20 μL. 

The minor modifications that we made from our previous method ^{11 12} included: transporting eyes on ice, perfusion through the ophthalmic artery with 140 mm Hg perfusion pressure as the cutoff point and a higher perfusion rate, measuring absorbance, and lengthening the time to obtain optimum flow rate to 1 hour. However, both methods produced similar results. 

Drugs and Chemicals

Acetazolamide, 3-(aminosulfonyl)-5-(butylamino)-4-phenoxybenzoic acid (bumetanide), 1β,3β,5β,11α,14,19-hexahydroxycard-20(22)-enolide3-(6-deoxy-α-l-mannopyranoside) (ouabain), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals, used in the Krebs’ solution and AH substitute, were of analytical grade and were obtained either from Sigma Chemical Co. or from Fisher Scientific Products (Pittsburgh, PA). Drugs were dissolved in DMSO before they were added to the perfusate or to the anterior chamber fluid, to obtain the intended final concentration. 

Statistical Analysis of Data

Results were expressed as the mean ± SEM of separate experiments. Statistical comparisons were made by two-tailed Student’s t-test for paired data and analysis of variance (ANOVA) followed by the Bonferroni post hoc multiple comparison tests. P <0.05 was considered significant. Graphs were drawn using a computer-based program (Prism; GraphPad, San Diego, CA). 

Results

Arterial Perfusion Pressure

The mean arterial perfusion pressures exhibited by different experimental groups of eyes were very similar and remained fairly steady throughout the experiment (Fig. 1) . The average pressure varied within the range of 34.13 ± 4.13 and 39.63 ± 6.17 mm Hg in different treatment groups of eyes. The mean pressure in all the eyes was 36.99 ± 1.42 mm Hg (n = 71). None of the drugs used in the present investigation had any significant effect on the arterial perfusion pressure of the in vitro eyes. 

Typical Experimental Records on AHF and IOP

Figures 2A and 2B show the typical experimental records on AHF and IOP, respectively. In both cases the slopes of the regression lines drawn on the natural logarithm (LN) of absorbance of fluorescein and IOP versus time before (control) and after (treated) addition of the drug were compared, to calculate statistical significance of differences. 

Effect of DMSO on AHF and IOP

To see whether the concentration of DMSO used in dissolving the drugs had any effect on AHF or IOP, we obtained and analyzed appropriate control data and found no effect (Tables 1 and 2) . Similarly, control experiments with saline had no effect on AHF or IOP (data not shown). 

Effect of Ouabain on AHF and IOP

We used the NaK-ATPase inhibitor ouabain to investigate its effect on in vitro AHF. Administration of 1.0 mM ouabain produced significant reduction in AHF when added to the stromal or aqueous side (Table 1) . When expressed in percentages, the reductions were 46% and 42%, respectively. After addition to both sides (1.0 mM), it produced a more pronounced effect on AHF (Table 1 ; 61% reduction). There was also a significant decrease in IOP from the respective control IOPs (Table 2) . The results suggest that most of the AH in the in vitro eye is formed by active ion transports dependent on the activity of NaK-ATPase. 

Effect of Bumetanide and Furosemide on AHF and IOP

Bumetanide (0.1 mM), a specific inhibitor of NKCC cotransport, and furosemide (0.1 mM), a nonspecific anion transport inhibitor, produced significant reduction of AHF when applied to the stromal side. These represent 35% reduction by bumetanide and 45% reduction by furosemide (Table 1) . A significant reduction in IOP was also observed in both cases of bumetanide and furosemide (Table 2) . 

Effects of DIDS and NPPB on AHF and IOP

DIDS (0.001–0.1 mM), which is believed to inhibit the Cl-HCO₃ exchanger, Na-HCO₃ cotransporter and chloride channel, produced a concentration-dependent reduction when added to the stromal side (Table 1) . The inhibition induced by the three concentrations used were 10%, 33%, and 55%, respectively. NPPB (0.1 mM), the chloride channel blocker at the nonpigmented cells, produced a significant reduction of AHF (25%) when applied to the aqueous side (Table 1) . DIDS similarly produced a significant and dose-dependent decrease in IOP with the two higher concentrations. NPPB also lowered IOP significantly (Table 2) . 

Effect of Acetazolamide on AHF and IOP

Stromal-side application of acetazolamide (0.1 mM) also significantly reduced both AHF (31%; Table 1 ) and IOP (Table 2) in the perfused eye. 

Discussion

In the present investigation, we used an in vitro whole-eye preparation to study the effects of known inhibitors of ion transport on AHF. We used this preparation because it allowed us to use drugs selectively on the stromal and/or aqueous side. The stromal side represents the PE cells and the aqueous side represents the NPE cells. We used ouabain, the selective inhibitor of NaK-ATPase; bumetanide, a specific inhibitor of NKCC cotransport; furosemide, a nonspecific inhibitor of anion transport; DIDS which is believed to inhibit the Cl-HCO₃ exchanger, Na-HCO₃ cotransporter and chloride channel; and NPPB, the chloride channel blocker in the nonpigmented cells; and acetazolamide, a carbonic anhydrase inhibitor (CAI). The present data support the conclusion that in the isolated bovine eye the AH is formed mostly by processes involving active secretion and chloride transport, which makes a significant contribution to active secretion. Active Cl⁻ secretion across the in vitro CE of cat, ⁷ toad, ¹³ rabbit, ^{5 6} and ox ^{9 14} has been demonstrated previously. The present finding is also consistent with these studies, indicating that anionic, specifically chloride, rather than cationic transport, is involved in the AHF. 

Ouabain

Ouabain is a specific blocker of NaK-ATPases and when applied to both PE and NPE cells produced a 61% decrease in AHF. When applied to either the PE or NPE side, it produced similar reductions in AHF by approximately 40% (Table 1) . 

NaK-ATPase is a primary active transport system that generates sodium gradients for other secondary active transport to take place. It has long been thought to be a prerequisite for AHF ¹⁵ and has been histochemically identified in the CE of rabbit ^{16 17} and ox. ¹⁸ NaK-ATPases were found mainly along the basolateral infoldings and interdigitations of both the PE and NPE cells ^{17 19} and higher activities and concentrations were found on the NPE cells. ^{2 17 20}  

Previously, it was thought that NPE cells were more important than the PE cells in the AHF. ²¹ The present result has shown that they are equally important in powering the AHF, because inhibiting the NaK-ATPases in NPE or PE cells produced similar effect. However, it has to be mentioned that the final concentration of ouabain on the aqueous side was not known when the drug was circulated to the anterior chamber. The concentration of ouabain in NPE cells depends on diffusion of drug to the posterior chamber (behind the iris) through the pupil. Because AH is continuously formed by the bovine ciliary body epithelium, the diffusion of ouabain (or other anterior chamber-administered drugs) must be against the direction of fluid flow. Therefore, it is highly plausible that the final concentration of the drug at the posterior chamber or NPE may not be the same as the calculated final concentration after dilution in the anterior circulation. Ouabain was administered into the anterior chamber by injecting highly concentrated stock solution into the lumen of the silicon tubing that comprised the anterior chamber’s circulating system. The drug was diluted as it was circulated through the anterior chamber and the final concentration was calculated according to the estimated total volume of the chamber circulation. Although the final concentration of ouabain in the posterior chamber was not known, it was clear that a significant inhibitory effect had been attained. Because strong inhibitory effects on the electrical parameters of bovine ciliary body epithelium were observed at a very low concentration of ouabain (0.01 mM), ²² a low final concentration of ouabain in the posterior chamber would have been sufficient to inhibit the NaK-ATPases in NPE cells significantly. 

According to the present data, the simplest interpretation would be that more than 60% of the AHF is due to an active- or energy-dependent process, and 40% is due to passive ultrafiltration. This finding lends support to our previous data ¹¹ that large changes in perfusion pressure had little effect on IOP and speculation that ultrafiltration plays a minor role in AHF in the perfused bovine eye. However, the present calculation may have underestimated the true contribution of active secretion, because when all the active transport process was halted with ouabain, the rate of AHF would be decreased and so would the IOP. This decrease in IOP may have favored more passive ultrafiltration of AH. Therefore, the resultant estimation of 40% ultrafiltration in AHF may be an overestimation. However, the exact contribution of a passive versus active process in the AHF is yet to be determined. Nevertheless, this inhibition with ouabain strongly suggests that AHF is predominantly driven by active process. ¹  

Bumetanide

Bumetanide is a specific inhibitor of NKCC cotransport. ²⁶ Its effect on the ion transport in CE has been intensely studied. A bumetanide-sensitive shrinkage of PE cells was observed. ²⁷ Bumetanide also decreased the short-circuited current (SCC) in bovine ciliary body epithelium in an Ussing chamber. ²² However, McLaughlin et al. ²⁵ found that bumetanide did not inhibit the intracellular Cl⁻ accumulation in isolated rabbit PE cells. They concluded that NKCC is not important in salt uptake by the PE cells. Later studies have found that bumetanide inhibits chloride secretion in rabbit ⁶ and bovine CE. ¹⁴ In addition, NKCC was found to be highly polarized at the basolateral membrane of bovine PE cells, ²⁰ which is consistent with its role in the vectorial transport of ions into the PE cells. 

The inhibitory effect of bumetanide on in vitro AHF in the present work was therefore not surprising. The 35% inhibition of the fluid formation in the whole-eye model suggests an important role for NKCC in AHF in the ox. Stromal application of bumetanide was found to decrease the SCC by 43% in rabbit ciliary body. ⁶ In addition, it inhibited by 54% the chloride secretion in bovine CE when applied to the stromal side. ¹⁴ The exact reason for a lower inhibition of fluid flow than chloride secretion or SCC by stromal bumetanide is unknown. It is also interesting to note that the theoretical prediction of fluid formation by chloride flux found in the ox is more than 10 times lower than that of the present system. ^{9 11} Therefore, there may be inherent differences between the present system (whole-eye preparation) and the Using-chamber system (dissected ciliary body preparation), in the fluid-formation mechanism. One of the major differences is that in the whole-eye system there is continuous perfusion or hydrostatic pressure in the ciliary stroma. Such pressure may either activate pump activities or simply unfold the ciliary processes in a way similar to in vivo anatomy. In addition, there could have been less traumatic damage to the tissues in the whole-eye preparation, which may have better preserved the complex transport activities of transporters and channels. 

Furosemide

To investigate whether AH flow with the whole-eye perfusion model depends on anionic transport, the effect of furosemide on the whole-eye system was examined. Furosemide is a nonspecific inhibitor that blocks anionic transport in many tissues. It inhibits the electrical parameters of isolated ciliary body of shark, ²⁶ rabbit, ²⁷ and ox. ²² It also abolishes chloride secretion by bovine CE. ²² In the present study, stromal furosemide inhibited 45.5% of in vitro AHF, which therefore suggests that the AHF in the whole-eye perfusion is at least in part driven by anionic transport, and chloride secretion is likely to be a major candidate. In addition to blocking the Na-K-2Cl cotransporter with a potency equal to that of bumetanide, ²⁸ additional inhibitory effects of furosemide on the K-Cl cotransporter, ^{29 30 31} and on the Cl⁻ channel ³² have been reported. This could explain the more pronounced effect of furosemide on AHF (45%) than bumetanide (35%) in the present investigation. The detailed involvement of anionic transport was studied, by using more specific blockers that have been known to act on candidate transporters and channel in the sodium chloride secretion across the CE. ^{6 14 25}  

DIDS

McLaughlin et al. ²⁵ proposed in their model of ion transport across the CE that the double or parallel exchangers are the key uptake pathway of sodium and chloride ions into the PE cells. The presence of both Cl⁻-CO₃ ⁻ and Na⁺-H⁺ exchangers have been suggested in cultured bovine PE cells. The Cl⁻-HCO₃ ⁻ exchanger may be responsible for chloride ion entry into the PE, ³³ whereas the Na⁺-H⁺ exchanger may mediate sodium entry into the PE. ³⁴ These exchangers are physically independent, but they are coupled physiologically by CA. ³⁵  

DIDS inhibits several transporters and channels in the CE, such as the Cl-HCO₃ exchanger, ³⁶ Na-HCO₃ cotransporter, ³⁷ and chloride channel. ³⁸ Its effect on the CE has been puzzling. Although it has been postulated to act on the Cl-HCO₃ exchanger at the PE cells, ²⁵ DIDS affects the SCC of monkey CE only when it is applied to the aqueous side but not the stromal side. ³⁹ It decreases the SCC in isolated CE preparations of rabbit ⁶ and ox. ¹⁴ However, it has no inhibitory effect on the chloride secretion across the isolated bovine CE preparation. ¹⁴ Therefore, the exact effect of DIDS on AHF is still unclear. 

Stromal perfusion of DIDS strongly inhibited (56%) in vitro AHF in the present system. This is in sharp contrast with the absence of effect on chloride secretion in isolated bovine CE tissue. The exact reason for this discrepancy is not clear. It may suggest that DIDS acts on an unknown nonchloride pathway that drives AHF, or there may be postmortem differences in the transport characteristics of isolated ciliary body epithelium and intact whole-eye system, in that the parallel exchangers were operating in the whole-eye system but not in isolated preparation. In the latter case, the data may reflect a major contribution of transporters such as the Cl-HCO₃ exchanger in the AHF, as suggested earlier. ²⁵ Further work is needed to identify the exact site of DIDS action. 

NPPB

Chloride in the CE bilayer is believed to leave the NPE cells through chloride channels ⁴ which constitutes the final path of transepithelial secretion of chloride into the anterior chamber. NPPB is a chloride channel inhibitor that has been shown to be effective in blocking chloride channels of the NPE cells. ⁴⁰ Using bovine pigmented and nonpigmented CE, Zhang and Jacob ⁴¹ have shown that NPPB prevents activation of all three Cl channels (low, intermediate, and high conductance/maxi). In other tissues, NPPB has been reported to inhibit potassium channels, ^{41 42 43 44} Cl-HCO₃ exchange, ⁴⁵ and gap junction hemichannels (connexin-50). ⁴⁶ NPPB has also been shown to uncouple oxidative phosphorylation by acting on mitochondrial anion channels. ⁴⁷ It has been suggested that the chloride channel is the rate-limiting step in chloride secretion across the CE. ⁴⁸ NPPB has also been shown to inhibit transepithelial chloride secretion by bovine ciliary body epithelium by 90%, when applied to the aqueous side. ¹⁴  

If chloride secretion is the major driving force for AHF, a strong inhibition of fluid flow would be expected if the chloride transport is abolished. However, it decreased the in vitro rate of AHF by only 25%. This result is even more surprising if we consider the inhibitory effects of NPPB in other tissues on K⁺ channels, Cl⁻-HCO₃ ⁻ exchange, gap junction protein, and oxidative phosphorylation. However, these additional mechanisms of NPPB are yet to be documented in CE cells. There are two possible reasons for this discrepancy. First, there may be other major ionic mechanisms operating in addition to chloride secretion. However, it has been shown that neither sodium ⁹ nor bicarbonate ⁴⁹ is actively secreted. Second, to act on the chloride channel, NPPB has to diffuse through the pupil to the posterior chamber. Because AH is continuously formed, this uphill diffusion of NPPB may be difficult and the final concentration is likely to be much less than the calculated value, a situation that was similar to the anterior ouabain application described earlier. In other words, the effect of NPPB may not have been optimal due to a low effective concentration in the NPE cells that were used. A higher concentration of NPPB was not used because more concentrated stock NPPB solution was difficult to prepare. 

Acetazolamide

Acetazolamide is a CAI that has been used clinically to lower the rate of AHF and IOP in glaucoma patients for several decades. ^{50 51} The presence of CA has been demonstrated by histochemical methods in the CE of the rabbit, the monkey, and humans. ^{52 53} Acetazolamide decreased the AHF in the whole-eye perfusion system by 31.4%. This is consistent with the notion that the CA enzyme in the CE is essential in bovine AHF. Clinically, acetazolamide also decreases IOP and aqueous flow by approximately 21% and 27%, respectively, which agrees well with the present in vitro observation. ⁵⁴ It also correlates well with an Ussing-chamber study in which acetazolamide decreased chloride transport by 21% when administered through the blood side of bovine CE. ⁴⁹ It has been postulated that CAI may work by supplying proton and bicarbonate ions to the parallel exchangers: Na-H and Cl-HCO₃. ^{49 55 56} Blockade of CA may result in a decrease in NaCl absorption through the parallel exchangers at the PE cells and thereby decrease chloride secretion and AHF. 

In conclusion, we have investigated the effects of a number of ion transport inhibitors on the in vitro rate of AHF. The results support a significant role of chloride transport in driving the active fluid formation by the CE. 

 Supported by UK/HK Joint Research Scheme Grant H-ZF36; the Croucher Foundation Grant H-ZH22; and Hong Kong Polytechnic University Grants G-V423 and G-T251.

 Submitted for publication April 19, 2002; revised September 24, 2002; accepted October 28, 2002.

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Figure 1.

 

Mean perfusion pressures of eyes in different treatment groups. The perfusion pressures were shown from 0 minutes (designated as the time point at which the anterior chamber started to be perfused with the fluorescein solution) to the end of the experiments. There was no significant change in perfusion pressures at any stage of the experiments with any of the drugs used. Routes of perfusion of drugs were as follows: ‡P, drug was added to the perfusate (i.e., to the stromal side); §AC, drug was added to the anterior chamber (to the aqueous humor); #P+AC, drug was added to both the stromal and the aqueous sides.

Figure 2.

 

Typical experimental records on AHF and IOP. (A) Changes in absorbance of fluorescein with time before and after addition of the drug. (B) Changes in IOP (mm Hg) with time before and after addition of the drug. Arrows: D and D1 indicate the time point of addition of drug to the perfusate or anterior chamber; T and T1 indicate starting point of collection of drug-treatment data, after a stabilization period of 20 minutes was allowed to establish the drug’s effect.

Table 1.

 

Effect of Drugs on AHF in the Isolated, Arterially Perfused Bovine Eye

Drug/Vehicle	Concentration (mM)	Route	AHF			n	P	Reduction (%)
									Control	Treated
DMSO	0.05%	P	43.0 ± 2.0	42.7 ± 2.0	6	NS	0.7
Ouabain	1.0	P	39.8 ± 3.8	21.2 ± 4.1	7	<0.05	46.7
Ouabain	1.0	AC	37.2 ± 3.8	21.3 ± 2.2	6	<0.01	42.7
Ouabain	1.0	P+AC	38.6 ± 3.6	14.9 ± 1.4	5	<0.01	61.4
Bumetanide	0.1	P	45.0 ± 2.4	29.8 ± 3.6	8	<0.001	35.1
Furosemide	0.1	P	41.3 ± 7.2	22.5 ± 4.7	9	<0.001	45.5
DIDS	0.001	P	47.0 ± 2.3	42.0 ± 5.3	8	NS	10.6
DIDS	0.01	P	48.0 ± 2.8	32.0 ± 2.5	7	<0.01	33.3
DIDS	0.1	P	45.0 ± 5.3	20.0 ± 5.4	6	>0.05	55.6
NPPB	0.1	AC	39.4 ± 3.2	29.7 ± 2.0	5	<0.01	24.6
Acetazolamide	0.1	P	35.0 ± 1.4	24.0 ± 2.5	10	<0.01	31.4

Table 2.

 

Effects of Drugs on IOP in the Isolated, Arterially Perfused Bovine Eye

Drug/Vehicle	Concentration (mM)	Route	IOP			n	P
								Control	Treated
DMSO	0.05%	P	−2.6 ± 0.4	−6.0 ± 2.0	6	NS
Ouabain	1.0	P	−1.2 ± 1.4	−30.0 ± 3.0	7	<0.001
Ouabain	1.0	AC	−2.2 ± 0.7	−28.0 ± 3.0	6	<0.001
Ouabain	1.0	P+AC	−2.3 ± 1.3	−50.0 ± 2.0	5	<0.001
Bumetanide	0.1	P	−2.0 ± 0.7	−24.0 ± 3.0	8	<0.01
Furosemide	0.1	P	−1.6 ± 0.6	−29.0 ± 5.0	9	<0.001
DIDS	0.001	P	−0.9 ± 0.6	−6.0 ± 2.0	8	NS
DIDS	0.01	P	0.2 ± 1.0	−21.0 ± 3.0	7	<0.001
DIDS	0.1	P	−1.1 ± 0.7	−41.0 ± 3.0	6	>0.001
NPPB	0.1	AC	−2.5 ± 1.8	−17.0 ± 1.0	5	<0.01
Acetazolamide	0.1	P	−1.7 ± 0.3	−18.0 ± 2.0	10	<0.001