pH Dependence of Corneal Oxygen Consumption - CiteSeerX
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IOVS, December 1998, Vol. 39, No. 13
pH Dependence of Corneal Oxygen Consumption Daniel M. Harvitt and Joseph A. Bonanno determine whether corneal acidosis, which occurs during contact lens wear, alters corneal O2 consumption (Qo2) and if so, whether increased ion transport activity could contribute to altered Qo2 during acidosis.
PURPOSE. TO
was measured, using the phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl) porphine, in an airtight chamber that held a trephined rabbit cornea. The rate of change in chamber Po2 was used as a measure of Qo2. Qo2 was measured at pH 7.5 and then at either pH 6.7, 7.1, or 7.3. Measurements of Qo2 at pHs 7.5 and 6.7 were repeated in the presence of 0.5 mM amiloride and 0.5 mM ouabain. METHODS. PO 2
When pH was changed from 7.5 to 6.7, 7.1, or 7.3, O2 consumption increased by a factor of 1.80 ±0.11 (±SE), 1.65 ± 0.12, and 1.44 ± 0.06, respectively. Amiloride (0.5 mM) and ouabain (0.5 mM) inhibited 50% and 65%, respectively, of the increase in Qo2 at pH 6.7.
RESULTS.
CONCLUSIONS. Corneal
acidosis leads to increased Qo2 in a dose-dependent manner. The increased Qo2 is in part secondary to the activation of pH regulator)' mechanisms, including Na + /H + exchange, which then stimulates Na + / K^-ATPase activity. These findings indicate that contact lens-induced acidosis can exacerbate corneal hypoxia and related complications. (Invest Ophthalmol Vis Sci. 1998; 39:2778-2781) a previous series of experiments, phosphorescence-based I nmeasurements of tear Po beneath contact lenses in the 2
rabbit were found to be significantly less than mathematical model predictions.' This model incorporates as its parameters the thickness and O2 permeability of the cornea, tears, and contact lens and the O2 consumption rate of the cornea (Qo2). Additionally, the endothelial and aqueous humor boundary conditions must be estimated. The most important parameter affecting the O2 distribution is Qo2. Varying the parameters of the model to determine the sensitivity of the tear Po2 estimate to errors in these parameter measurements,2 and examining the methodology of these measures, led to a reexamination of
From the Vision Science Group and the Morton D. Sarver Center for Cornea and Contact Lens Research, University of California, School of Optometry, Berkeley, California. Supported by the Morton D. Sarver Center, University of California, School of Optometry, Berkeley, California; the American Optometric Foundation, Sigma Xi, and the National Institutes of Health, Bethesda, Maryland (Grants T32 EY07043 and EY08834). Portions of this work were presented in abstract form at the 1996 and 1997 Annual Meetings of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida. Submitted for publication April 6, 1998; revised August 6, 1998; accepted August 19, 1998. Proprietary interest category: N. Reprint requests: Joseph Bonanno, Indiana University, School of Optometry, 88 East Atwater Avenue, Bloomington, IN 47405.
the Qo2 rates.3 However, these Qo2 measurements were not significantly different from previous estimations, and therefore did not explain the discrepancy between our direct tear O2 measurements and the model predictions. One important difference between the in vivo tear O2 measurements and the conditions of the in vitro Qo2 measurements is pH. During contact lens wear, the cornea is acidified by anaerobic metabolic products and CO2 retention. Contact lens wear acidifies corneal epithelium,4 stroma,5 endothelium,6 aqueous humor,4 and tears.7 The extent of acidification is inversely related to the O2 transmissibility of the lens. Changes in intracellular pH (pH;) can have significant effects on cellular physiology, including alterations in glycolytic activity, ion transport activity, and mitochondrial respiration, all of which could affect Qo2 during contact lens wear. In particular, it is known that acidosis activates pH regulatory mechanisms such as Na + /H + exchange,8'9 which results in an increase in intracellular Na+. In the amphibian cornea, increased intracellular Na+ results in increased Na+/K+-ATPase activity and Qo2.10 Therefore, the goal of this study was to determine whether corneal acidosis alters Qo2 and if acidosis does influence Qo2, whether it invokes increased transport activity due to pH; regulation?
MATERIALS AND METHODS Chemicals and Solutions The O2-sensitive phosphorescent dye Pd meso-tetra (4-carboxyphenyl) porphine (Porphyrin Products, Logan, UT) was used in all the experiments. Porphine (20 JLIM) and bovine serum albumin (0.04%; Sigma, St. Louis, MO) were dissolved in Ringer's solution consisting of (in mM) 115 NaCl, 2 K2HPO4, 0.61 MgCl2, 1.4 Ca+-gluconate, and 28.5 Na+-gluconate, 20.0 HEPES, pH 7.50 ± 0.02, with osmolarity adjusted to 300 ± 5 mOsm with sucrose. Solution pH was varied by adding HCl and NaOH. Amiloride and ouabain were obtained from Sigma. Solutions were filtered using a sterile 0.2-/u,m low-protein-binding single-use filter (Acrodisc; Gelman Sciences, Ann Arbor, MI). Procedure Measurement of rabbit Qo2 was described previously.3 Trephined corneas were preincubated with drugs or new solution pH for 20 to 30 minutes before measurements were made. In all experiments, repeated series of measurements were made at each pH level. These repeated measurements (see Fig. 1) were intended to ensure that equilibrium had been reached. If consecutive series of measurements under identical conditions yielded significantly different O2 consumption rates, then another series of measurements was taken. After measurement of the O2 consumption rates at pH 7.5 and either pH 6.7 or 7.1, the chamber was refilled with pH 7.5 solution to examine recovery of O2 consumption to the initial rate. Previous studies using the porphine dyes" have shown that the phosphorescence quenching constants for O2 are unaffected by pH over the range being used in these experiments. To test the effects of amiloride (an Na + /H + exchange inhibitor) and ouabain (an Na+/K+-ATPase inhibitor) on O2 metabolism under acidic conditions, the O2 consumption rate was first measured at pH 7.5. Next the consumption rate was
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Time (minutes) FIGURE 1. Corneal chamber Po2 versus time. (A) Rabbit cornea at pHs 7.5 and 6.7. The cornea was incubated in each solution for 20 minutes before the series of measurements was started. The cornea was initially placed in pH 7.5 solution (circles), then in pH 6.7 (triangles), and then returned to pH 7.5 (squares). In this experiment, O2 consumption increased 87% in pH 6.7 compared with pH 7.5. When the cornea was returned to pH 7.5 solution, there was incomplete recovery toward the initial O2 consumption rate, and corneal O2 consumption was 27% higher than during the first series of measurements. (B) Rabbit cornea at pHs 7.5 (circles) and 7.3 (triangles and diamonds). The steeper slope of the chamber Po2 versus time regression at pH 7.3 indicates a 24% increase in the O2 metabolic rate for this cornea.
measured at pH 7.5 with the drug, and then at pH 6.7 with the drug. The amount of increased Qo2 at pH 6.7 compared with pH 7.5 in the presence of drugs was compared with the previous experiments in which Qo2 was measured with no drugs at these pH levels.
Statistics and Calculations Change in chamber Po2 per unit time (millimeters of Hg per minute) was used as the expression of O2 consumption (Qo2). The ratios of O2 consumption rates at different pHs are ex-
2780 TABLE
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JOVS, December 1998, Vol. 39, No. 13
1. Change in Chamber Po2 per Minute for Rabbit Corneas in pH 7.5 and 6.7
A(Po 2 /min)
Qo2 pH 7.5a
Qo2 pH 6.7
Qo 2 pH 7.5b
Qo2 ratio pH 6.7/7.5
Qo2 ratio pH 7.5b/7.5a
2.25 ± 0.23
3.93 ± 0.36
2.91 ± 0.24
1.80 ± 0.11*
1.35 ± 0.09*
Values are means ± SE; n = 10. Qo2, corneal O2 consumption rate. *P < 0.05. pressed as the average of the ratio from individual experiments, such that
for n experiments. Two-sample Student's /-test assuming unequal variances was performed to test whether the change in Qo2 from pH 7 5 to pH 6.7 was significantly different when performed in the presence or absence of the transport inhibitors ouabain and amiloride. RESULTS
Figure 1A shows a typical change in chamber Po2 over time for a cornea bathed at pH 7.5, 6.7, and then again at 7.5. The rate of change in chamber Po2, indicated by the slope of the data, is higher at pH 6.7. Table 1 summarizes the results; the pHs are shown as pH 7.5a, pH 6.7, and pH 7.5b, respectively. Data are expressed as average change in chamber Po2 (in millimeters of Hg) per minute. Under the reduced pH conditions, Qo2 was found to increase dramatically, by a factor of 1.80 above the measured rate at pH 7.5 (Fig. 1, pH 6.7/7.5 column in Table 1). When returning to pH 7.5 after exposure to the acidic solution, the O2 consumption rate decreased toward the initial rate in pH 7.5. However, there was incomplete recovery because the consumption rate was on average 1.35 times the initial rate at pH 7.5 (Fig. 1, pH 7.5b/7.5a column in Table 1). Qo2 rates were also measured in experiments in which the cornea was initially in the control solution of pH 7.5 and then at intermediate pH levels of 7.1 or 7.3. Figure IB shows a typical response at pH 7.3 in which the increased O2 consumption was more subtle. Figure 2 shows that O2 consumption increases with acidosis in a dose-dependent manner. When pH was changed from 7.5 to 6.7, 7.1, and 7.3, O2 consumption increased by a factor of 1.80 ± 0.11 (±SE), 1.65 ± 0.12, and 1.44 ± 0.06, respectively. To examine the basis for the increased Qo2 with acidosis, similar experiments were performed in the presence of amiloride and ouabain. Qo2 was measured as in the earlier experiments in pH 7.5 solution. The consumption rate was then measured at pH 7.5 with the drug and then at pH 6.7 with the drug. Table 2 summarizes these experiments. With 0.5 mM amiloride, no inhibition of O2 metabolism was measured at pH 75, but Qo2 increased by a factor of 1.40 in pH 6.7 solution. Thus, amiloride inhibited 50% of the increase in Qo2. Similar results were observed with 1.0 mM amiloride (data not shown). In the presence of 0.5 mM ouabain, no inhibition of O2 metabolism was measured at pH 7.5, but Qo| increased by a factor of 1.28 in pH 6.7 solution. Ouabain therefore inhibited 65% of the increase in Qo2.
DISCUSSION The goal of the experiments described here was to investigate the effect of contact lens-induced corneal acidosis on O2 metabolism. Excised corneas were placed in an airtight chamber, and the Qo2 was calculated from the change in Po2 of the stirred chamber solution over time. Epithelial Qo2 is likely to have a more dominant role in our Qo2 measurements because it was measured in the absence of HCO3~, which will suppress endothelial metabolism. HCO3~-free conditions were used to facilitate control of pH. Qo2 was measured at pH 7.5 and then at pH 7.3, 7.1, or 6.7; there were dose-dependent increases in Qo2 of 44%, 65%, and 80%, respectively (Table 1, Fig. 2). We hypothesized that the increased Qo2 was being caused by the energy requirements of pH; regulation. Acidosis activates Na + /H + exchange,8'9 resulting in increased intracellular Na+, which in turn causes an increase in Na+/K+-ATPase activity. Hydrolysis of ATP and the reduction of the cellular energy state stimulates oxidative phosphorylation, increased O2 consumption, and glycolysis. According to this hypothesis, the increased Qo2 rate with acidosis should be inhibited by amiloride, an Na + /H + exchange inhibitor, and by ouabain, an inhibitor of Na+/K+-ATPase, as seen in the frog cornea after increased Na+ uptake.10 Indeed, amiloride reduced the increased Qo2 by 50% and ouabain by 65% (Table 2). Qo2 was measured previously as a function of pH primarily to examine the effects of acid and alkali burns. 1213 In each of these studies the cornea was exposed to short-duration acidic load, and then Qo2 was measured following the removal of the bathing acid solution. These experiments indicated that an increased Qo2 rate was present with acidosis; however, the
2.0 -r
1.0
7.5
7.4
7.3
7.2
7.1
6.9
6.8
6.7
pH 2. Increase in corneal O2 consumption (Qo2) under acidic conditions as a function of pH. The increase in consumption is expressed relative to the Qo2 at pH 7.5. Number of experiments at each pH is indicated in parentheses. Error bars, SE.
FIGURE
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IOVS, December 1998, Vol. 39, No. 13 TABLE 2 .
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Effect of Transport ][nhibitors on O 2 Consumption Po 2: a t pHs 7.5 and 6.7
Drug Control in = 15) 0.5 mM Amiloride (n = 6) 0.5 mM Ouabain (n = 9)
PH 7.5
PH 7.5 + drug
pH 6.7 + drug
(pH 7.5 + drug)/ PH7.5
(pH 6.7 + drug)/ (pH 7.5 + drug)
2 .25 ± 0.23 2 .30 ± 0.24 2 .26 ± 0.21
2.32 ± 0.18 2.41 ± 0.28
3.93 ±0.36 3.18 ± 0.15 3.07 ±0.36
1.03 ± 0.05 1.06 ± 0.07
1.80 ± 0.11 1.40 ± 0.09* 1.28 ± 0.09*
Values are means ± SE, expressed as APo2/min. *P < 0.05.
effect was small because of the short duration of the acidic stimulus. In the present study, we reduced corneal pH by bathing corneas in a reduced-pH Ringer's solution. Although we did not measure pHi; it has been shown that changes in extracellular pH in the cornea and most cell types lead to similar changes in pH,. l 4 1 5 When chamber Po2 measurements were made immediately after a change to acidic solution, no change in Qo2 was detectable (data not shown). However, repeated measures after 20 minutes of incubation indicated that a new steady state Qo2 had been reached. Presumably, this is because of the time required to reach a new steady state pHj. On returning corneas from the pH 6.7 condition to pH 7.5, there was incomplete recovery of Qo2 (Fig. 1, pH 7.5b/7.5a column, Table 1). The cause was not pursued, but it is possible that when pH is returned to pH 7.5 after pH 6.7, the pH 7.5 condition represents an alkalinizing load. The cornea would then have to activate acidifying mechanisms (e.g., C1~/HCO3~ and K + /H + exchange), which, like the alkalinizing Na + /H + exchange, require a net increase in ATP utilization and Qo2. During in vivo measurements of epithelial acidification during contact lens wear in rabbits, Giasson and Bonanno4 found that pH immediately after contact lens removal quickly overshoots the baseline pH level (i.e., an alkaline load) and then slowly returns over 30 minutes. This would help explain a delay in returning to the initial Qo2 rate. Ouabain and amiloride had no effect on Qo2 at pH 7.5 over 2 hours, consistent with the very limited activity of the Na + /H + exchanger at resting pH.9 Furthermore, other studies have shown that under control conditions ouabain had no effect on glycolytic activity16 or the metabolic ratio of reduced pyridine nucleotides to oxidized flavoproteins in endothelium.17 In the frog cornea, however, ouabain has inhibitory effects on Qo2 at pH 8.6.' 8 Thus, it may be that Na^/H4" exchange is active in frog corneas under resting conditions. The experiments presented in this article demonstrate that there is an increase in O2 consumption with acidosis, and experiments with transport inhibitors support the hypothesis that the increased O2 consumption is in part secondary to activated Na^/H4" exchange. If the changes in Qo2 with acidosis described here occur with contact lens wear, it may be possible to explain the difference between direct tear Po2 measurements beneath contact lenses and model predictions. 119 Further analysis of the model using these new Qo2 values are needed to address this question.
References 1. Harvitt DM, Bonanno JA. Direct noninvasive measurement of tear oxygen tension beneath gas-permeable contact lenses in rabbits. Invest Opbthalmol Vis Set. 1996;37:1026 -1036. 2. Lin DB. Oxygen Supply to the Cornea of an Open and Closed Eye Wearing a Contact Lens. Berkeley: University of California at Berkeley; 1992. 3. Harvitt DM, Bonanno JA. Oxygen consumption of the rabbit cornea. Invest Ophthalmol Vis Sci. 1998;39:444-448. 4. Giasson CJ, Bonanno JA. Corneal epithelial and aqueous humor acidification during in vivo contact lens wear in rabbits. Invest Opbthalmol Vis Sci. 1994;35:851-86l. 5. Bonanno JA, Poise KA. Effect of rigid contact lens oxygen transmissibility on stromal pH in the living human eye. Ophthalmology. 1987;94:13O5-13O9. 6. Giasson C, Bonanno J. Facilitated transport of lactate by rabbit corneal endothelium. Exp Eye Res. 1994;59:73-81. 7. Chen FS, Maurice DM. The pH in the precorneal tear film and under a contact lens measured with a fluorescent probe. Exp Eye Res. 199O;5O:251-259. 8. Bonanno JA, Giasson CJ. Intracellular pH regulation in fresh and cultured bovine endothelium, I: Na+/H+ exchange in the absence and presence of HCO3~. Invest Ophthalmol Vis Sci. 1992;33: 3058-3067. 9. Bonanno JA, Machen TE. Intracellular pH regulation in basal corneal epithelial cells measured in corneal explants: characterization of Na/H exchange. Exp Eye Res. 1989;49:129-l42. 10. Candia OA, Bentley PJ, Cook PI. Stimulation by amphotericin B of active Na transport across amphibian cornea. Am J Physiol. 1974; 226:1438-1444. 11. Shonat RD, Wilson DF, Riva CE, Pawlowski M. Oxygen distribution in the retinal and choroidal vessels of the cat as measured by a new phosphorescence imaging method. Appl Optics. 1992;31:37113718. 12. Carney LG, Efron N. pH ambiant et flux d'oxygene corneen.y Fr Ophtalmol, 1980;3:125-126. 13- Flynn WJ, Mauger TF. Corneal burns: a quantitative comparison of acid and base. Acta Opbthalmol. 1984;62:542-548. 14. Roos A, Boron WF. Intracellular pH. Physiol Rev. 1981 ;61:296 434. 15. Bonanno JA. Regulation of corneal epithelial intracellular pH. Optorn Vis Sci. 1991;68:582-586. 16. Riley M, Winkler B. Strong Pasteur effect in rabbit corneal endothelium preserves fluid transport under anaerobic conditions. J Physiol, 1990;426:81-93. 17. Laing R, Chiba K, Tsubota K, Oak S. Metabolic and morphologic changes in the corneal endothelium. Invest Ophthalmol Vis Sci. 1992;33:3315-3324. 18. Candia OA, Reinach PS. Thermodynamic analysis of active sodium and potassium transport in the frog corneal epithelium. Am J Physiol. 1982;242:F690-F698. 19. Fatt I, Bieber MT, Pye SD. Steady state distribution of oxygen and carbon dioxide in the in vivo cornea of an eye covered by a gas-permeable contact lens. AmJ Optom. 1969;46:3-l4.
Reports
IOVS, December 1998, Vol. 39, No. 13
pH Dependence of Corneal Oxygen Consumption Daniel M. Harvitt and Joseph A. Bonanno determine whether corneal acidosis, which occurs during contact lens wear, alters corneal O2 consumption (Qo2) and if so, whether increased ion transport activity could contribute to altered Qo2 during acidosis.
PURPOSE. TO
was measured, using the phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl) porphine, in an airtight chamber that held a trephined rabbit cornea. The rate of change in chamber Po2 was used as a measure of Qo2. Qo2 was measured at pH 7.5 and then at either pH 6.7, 7.1, or 7.3. Measurements of Qo2 at pHs 7.5 and 6.7 were repeated in the presence of 0.5 mM amiloride and 0.5 mM ouabain. METHODS. PO 2
When pH was changed from 7.5 to 6.7, 7.1, or 7.3, O2 consumption increased by a factor of 1.80 ±0.11 (±SE), 1.65 ± 0.12, and 1.44 ± 0.06, respectively. Amiloride (0.5 mM) and ouabain (0.5 mM) inhibited 50% and 65%, respectively, of the increase in Qo2 at pH 6.7.
RESULTS.
CONCLUSIONS. Corneal
acidosis leads to increased Qo2 in a dose-dependent manner. The increased Qo2 is in part secondary to the activation of pH regulator)' mechanisms, including Na + /H + exchange, which then stimulates Na + / K^-ATPase activity. These findings indicate that contact lens-induced acidosis can exacerbate corneal hypoxia and related complications. (Invest Ophthalmol Vis Sci. 1998; 39:2778-2781) a previous series of experiments, phosphorescence-based I nmeasurements of tear Po beneath contact lenses in the 2
rabbit were found to be significantly less than mathematical model predictions.' This model incorporates as its parameters the thickness and O2 permeability of the cornea, tears, and contact lens and the O2 consumption rate of the cornea (Qo2). Additionally, the endothelial and aqueous humor boundary conditions must be estimated. The most important parameter affecting the O2 distribution is Qo2. Varying the parameters of the model to determine the sensitivity of the tear Po2 estimate to errors in these parameter measurements,2 and examining the methodology of these measures, led to a reexamination of
From the Vision Science Group and the Morton D. Sarver Center for Cornea and Contact Lens Research, University of California, School of Optometry, Berkeley, California. Supported by the Morton D. Sarver Center, University of California, School of Optometry, Berkeley, California; the American Optometric Foundation, Sigma Xi, and the National Institutes of Health, Bethesda, Maryland (Grants T32 EY07043 and EY08834). Portions of this work were presented in abstract form at the 1996 and 1997 Annual Meetings of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida. Submitted for publication April 6, 1998; revised August 6, 1998; accepted August 19, 1998. Proprietary interest category: N. Reprint requests: Joseph Bonanno, Indiana University, School of Optometry, 88 East Atwater Avenue, Bloomington, IN 47405.
the Qo2 rates.3 However, these Qo2 measurements were not significantly different from previous estimations, and therefore did not explain the discrepancy between our direct tear O2 measurements and the model predictions. One important difference between the in vivo tear O2 measurements and the conditions of the in vitro Qo2 measurements is pH. During contact lens wear, the cornea is acidified by anaerobic metabolic products and CO2 retention. Contact lens wear acidifies corneal epithelium,4 stroma,5 endothelium,6 aqueous humor,4 and tears.7 The extent of acidification is inversely related to the O2 transmissibility of the lens. Changes in intracellular pH (pH;) can have significant effects on cellular physiology, including alterations in glycolytic activity, ion transport activity, and mitochondrial respiration, all of which could affect Qo2 during contact lens wear. In particular, it is known that acidosis activates pH regulatory mechanisms such as Na + /H + exchange,8'9 which results in an increase in intracellular Na+. In the amphibian cornea, increased intracellular Na+ results in increased Na+/K+-ATPase activity and Qo2.10 Therefore, the goal of this study was to determine whether corneal acidosis alters Qo2 and if acidosis does influence Qo2, whether it invokes increased transport activity due to pH; regulation?
MATERIALS AND METHODS Chemicals and Solutions The O2-sensitive phosphorescent dye Pd meso-tetra (4-carboxyphenyl) porphine (Porphyrin Products, Logan, UT) was used in all the experiments. Porphine (20 JLIM) and bovine serum albumin (0.04%; Sigma, St. Louis, MO) were dissolved in Ringer's solution consisting of (in mM) 115 NaCl, 2 K2HPO4, 0.61 MgCl2, 1.4 Ca+-gluconate, and 28.5 Na+-gluconate, 20.0 HEPES, pH 7.50 ± 0.02, with osmolarity adjusted to 300 ± 5 mOsm with sucrose. Solution pH was varied by adding HCl and NaOH. Amiloride and ouabain were obtained from Sigma. Solutions were filtered using a sterile 0.2-/u,m low-protein-binding single-use filter (Acrodisc; Gelman Sciences, Ann Arbor, MI). Procedure Measurement of rabbit Qo2 was described previously.3 Trephined corneas were preincubated with drugs or new solution pH for 20 to 30 minutes before measurements were made. In all experiments, repeated series of measurements were made at each pH level. These repeated measurements (see Fig. 1) were intended to ensure that equilibrium had been reached. If consecutive series of measurements under identical conditions yielded significantly different O2 consumption rates, then another series of measurements was taken. After measurement of the O2 consumption rates at pH 7.5 and either pH 6.7 or 7.1, the chamber was refilled with pH 7.5 solution to examine recovery of O2 consumption to the initial rate. Previous studies using the porphine dyes" have shown that the phosphorescence quenching constants for O2 are unaffected by pH over the range being used in these experiments. To test the effects of amiloride (an Na + /H + exchange inhibitor) and ouabain (an Na+/K+-ATPase inhibitor) on O2 metabolism under acidic conditions, the O2 consumption rate was first measured at pH 7.5. Next the consumption rate was
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Time (minutes) FIGURE 1. Corneal chamber Po2 versus time. (A) Rabbit cornea at pHs 7.5 and 6.7. The cornea was incubated in each solution for 20 minutes before the series of measurements was started. The cornea was initially placed in pH 7.5 solution (circles), then in pH 6.7 (triangles), and then returned to pH 7.5 (squares). In this experiment, O2 consumption increased 87% in pH 6.7 compared with pH 7.5. When the cornea was returned to pH 7.5 solution, there was incomplete recovery toward the initial O2 consumption rate, and corneal O2 consumption was 27% higher than during the first series of measurements. (B) Rabbit cornea at pHs 7.5 (circles) and 7.3 (triangles and diamonds). The steeper slope of the chamber Po2 versus time regression at pH 7.3 indicates a 24% increase in the O2 metabolic rate for this cornea.
measured at pH 7.5 with the drug, and then at pH 6.7 with the drug. The amount of increased Qo2 at pH 6.7 compared with pH 7.5 in the presence of drugs was compared with the previous experiments in which Qo2 was measured with no drugs at these pH levels.
Statistics and Calculations Change in chamber Po2 per unit time (millimeters of Hg per minute) was used as the expression of O2 consumption (Qo2). The ratios of O2 consumption rates at different pHs are ex-
2780 TABLE
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JOVS, December 1998, Vol. 39, No. 13
1. Change in Chamber Po2 per Minute for Rabbit Corneas in pH 7.5 and 6.7
A(Po 2 /min)
Qo2 pH 7.5a
Qo2 pH 6.7
Qo 2 pH 7.5b
Qo2 ratio pH 6.7/7.5
Qo2 ratio pH 7.5b/7.5a
2.25 ± 0.23
3.93 ± 0.36
2.91 ± 0.24
1.80 ± 0.11*
1.35 ± 0.09*
Values are means ± SE; n = 10. Qo2, corneal O2 consumption rate. *P < 0.05. pressed as the average of the ratio from individual experiments, such that
for n experiments. Two-sample Student's /-test assuming unequal variances was performed to test whether the change in Qo2 from pH 7 5 to pH 6.7 was significantly different when performed in the presence or absence of the transport inhibitors ouabain and amiloride. RESULTS
Figure 1A shows a typical change in chamber Po2 over time for a cornea bathed at pH 7.5, 6.7, and then again at 7.5. The rate of change in chamber Po2, indicated by the slope of the data, is higher at pH 6.7. Table 1 summarizes the results; the pHs are shown as pH 7.5a, pH 6.7, and pH 7.5b, respectively. Data are expressed as average change in chamber Po2 (in millimeters of Hg) per minute. Under the reduced pH conditions, Qo2 was found to increase dramatically, by a factor of 1.80 above the measured rate at pH 7.5 (Fig. 1, pH 6.7/7.5 column in Table 1). When returning to pH 7.5 after exposure to the acidic solution, the O2 consumption rate decreased toward the initial rate in pH 7.5. However, there was incomplete recovery because the consumption rate was on average 1.35 times the initial rate at pH 7.5 (Fig. 1, pH 7.5b/7.5a column in Table 1). Qo2 rates were also measured in experiments in which the cornea was initially in the control solution of pH 7.5 and then at intermediate pH levels of 7.1 or 7.3. Figure IB shows a typical response at pH 7.3 in which the increased O2 consumption was more subtle. Figure 2 shows that O2 consumption increases with acidosis in a dose-dependent manner. When pH was changed from 7.5 to 6.7, 7.1, and 7.3, O2 consumption increased by a factor of 1.80 ± 0.11 (±SE), 1.65 ± 0.12, and 1.44 ± 0.06, respectively. To examine the basis for the increased Qo2 with acidosis, similar experiments were performed in the presence of amiloride and ouabain. Qo2 was measured as in the earlier experiments in pH 7.5 solution. The consumption rate was then measured at pH 7.5 with the drug and then at pH 6.7 with the drug. Table 2 summarizes these experiments. With 0.5 mM amiloride, no inhibition of O2 metabolism was measured at pH 75, but Qo2 increased by a factor of 1.40 in pH 6.7 solution. Thus, amiloride inhibited 50% of the increase in Qo2. Similar results were observed with 1.0 mM amiloride (data not shown). In the presence of 0.5 mM ouabain, no inhibition of O2 metabolism was measured at pH 7.5, but Qo| increased by a factor of 1.28 in pH 6.7 solution. Ouabain therefore inhibited 65% of the increase in Qo2.
DISCUSSION The goal of the experiments described here was to investigate the effect of contact lens-induced corneal acidosis on O2 metabolism. Excised corneas were placed in an airtight chamber, and the Qo2 was calculated from the change in Po2 of the stirred chamber solution over time. Epithelial Qo2 is likely to have a more dominant role in our Qo2 measurements because it was measured in the absence of HCO3~, which will suppress endothelial metabolism. HCO3~-free conditions were used to facilitate control of pH. Qo2 was measured at pH 7.5 and then at pH 7.3, 7.1, or 6.7; there were dose-dependent increases in Qo2 of 44%, 65%, and 80%, respectively (Table 1, Fig. 2). We hypothesized that the increased Qo2 was being caused by the energy requirements of pH; regulation. Acidosis activates Na + /H + exchange,8'9 resulting in increased intracellular Na+, which in turn causes an increase in Na+/K+-ATPase activity. Hydrolysis of ATP and the reduction of the cellular energy state stimulates oxidative phosphorylation, increased O2 consumption, and glycolysis. According to this hypothesis, the increased Qo2 rate with acidosis should be inhibited by amiloride, an Na + /H + exchange inhibitor, and by ouabain, an inhibitor of Na+/K+-ATPase, as seen in the frog cornea after increased Na+ uptake.10 Indeed, amiloride reduced the increased Qo2 by 50% and ouabain by 65% (Table 2). Qo2 was measured previously as a function of pH primarily to examine the effects of acid and alkali burns. 1213 In each of these studies the cornea was exposed to short-duration acidic load, and then Qo2 was measured following the removal of the bathing acid solution. These experiments indicated that an increased Qo2 rate was present with acidosis; however, the
2.0 -r
1.0
7.5
7.4
7.3
7.2
7.1
6.9
6.8
6.7
pH 2. Increase in corneal O2 consumption (Qo2) under acidic conditions as a function of pH. The increase in consumption is expressed relative to the Qo2 at pH 7.5. Number of experiments at each pH is indicated in parentheses. Error bars, SE.
FIGURE
Reports
IOVS, December 1998, Vol. 39, No. 13 TABLE 2 .
2781
Effect of Transport ][nhibitors on O 2 Consumption Po 2: a t pHs 7.5 and 6.7
Drug Control in = 15) 0.5 mM Amiloride (n = 6) 0.5 mM Ouabain (n = 9)
PH 7.5
PH 7.5 + drug
pH 6.7 + drug
(pH 7.5 + drug)/ PH7.5
(pH 6.7 + drug)/ (pH 7.5 + drug)
2 .25 ± 0.23 2 .30 ± 0.24 2 .26 ± 0.21
2.32 ± 0.18 2.41 ± 0.28
3.93 ±0.36 3.18 ± 0.15 3.07 ±0.36
1.03 ± 0.05 1.06 ± 0.07
1.80 ± 0.11 1.40 ± 0.09* 1.28 ± 0.09*
Values are means ± SE, expressed as APo2/min. *P < 0.05.
effect was small because of the short duration of the acidic stimulus. In the present study, we reduced corneal pH by bathing corneas in a reduced-pH Ringer's solution. Although we did not measure pHi; it has been shown that changes in extracellular pH in the cornea and most cell types lead to similar changes in pH,. l 4 1 5 When chamber Po2 measurements were made immediately after a change to acidic solution, no change in Qo2 was detectable (data not shown). However, repeated measures after 20 minutes of incubation indicated that a new steady state Qo2 had been reached. Presumably, this is because of the time required to reach a new steady state pHj. On returning corneas from the pH 6.7 condition to pH 7.5, there was incomplete recovery of Qo2 (Fig. 1, pH 7.5b/7.5a column, Table 1). The cause was not pursued, but it is possible that when pH is returned to pH 7.5 after pH 6.7, the pH 7.5 condition represents an alkalinizing load. The cornea would then have to activate acidifying mechanisms (e.g., C1~/HCO3~ and K + /H + exchange), which, like the alkalinizing Na + /H + exchange, require a net increase in ATP utilization and Qo2. During in vivo measurements of epithelial acidification during contact lens wear in rabbits, Giasson and Bonanno4 found that pH immediately after contact lens removal quickly overshoots the baseline pH level (i.e., an alkaline load) and then slowly returns over 30 minutes. This would help explain a delay in returning to the initial Qo2 rate. Ouabain and amiloride had no effect on Qo2 at pH 7.5 over 2 hours, consistent with the very limited activity of the Na + /H + exchanger at resting pH.9 Furthermore, other studies have shown that under control conditions ouabain had no effect on glycolytic activity16 or the metabolic ratio of reduced pyridine nucleotides to oxidized flavoproteins in endothelium.17 In the frog cornea, however, ouabain has inhibitory effects on Qo2 at pH 8.6.' 8 Thus, it may be that Na^/H4" exchange is active in frog corneas under resting conditions. The experiments presented in this article demonstrate that there is an increase in O2 consumption with acidosis, and experiments with transport inhibitors support the hypothesis that the increased O2 consumption is in part secondary to activated Na^/H4" exchange. If the changes in Qo2 with acidosis described here occur with contact lens wear, it may be possible to explain the difference between direct tear Po2 measurements beneath contact lenses and model predictions. 119 Further analysis of the model using these new Qo2 values are needed to address this question.
References 1. Harvitt DM, Bonanno JA. Direct noninvasive measurement of tear oxygen tension beneath gas-permeable contact lenses in rabbits. Invest Opbthalmol Vis Set. 1996;37:1026 -1036. 2. Lin DB. Oxygen Supply to the Cornea of an Open and Closed Eye Wearing a Contact Lens. Berkeley: University of California at Berkeley; 1992. 3. Harvitt DM, Bonanno JA. Oxygen consumption of the rabbit cornea. Invest Ophthalmol Vis Sci. 1998;39:444-448. 4. Giasson CJ, Bonanno JA. Corneal epithelial and aqueous humor acidification during in vivo contact lens wear in rabbits. Invest Opbthalmol Vis Sci. 1994;35:851-86l. 5. Bonanno JA, Poise KA. Effect of rigid contact lens oxygen transmissibility on stromal pH in the living human eye. Ophthalmology. 1987;94:13O5-13O9. 6. Giasson C, Bonanno J. Facilitated transport of lactate by rabbit corneal endothelium. Exp Eye Res. 1994;59:73-81. 7. Chen FS, Maurice DM. The pH in the precorneal tear film and under a contact lens measured with a fluorescent probe. Exp Eye Res. 199O;5O:251-259. 8. Bonanno JA, Giasson CJ. Intracellular pH regulation in fresh and cultured bovine endothelium, I: Na+/H+ exchange in the absence and presence of HCO3~. Invest Ophthalmol Vis Sci. 1992;33: 3058-3067. 9. Bonanno JA, Machen TE. Intracellular pH regulation in basal corneal epithelial cells measured in corneal explants: characterization of Na/H exchange. Exp Eye Res. 1989;49:129-l42. 10. Candia OA, Bentley PJ, Cook PI. Stimulation by amphotericin B of active Na transport across amphibian cornea. Am J Physiol. 1974; 226:1438-1444. 11. Shonat RD, Wilson DF, Riva CE, Pawlowski M. Oxygen distribution in the retinal and choroidal vessels of the cat as measured by a new phosphorescence imaging method. Appl Optics. 1992;31:37113718. 12. Carney LG, Efron N. pH ambiant et flux d'oxygene corneen.y Fr Ophtalmol, 1980;3:125-126. 13- Flynn WJ, Mauger TF. Corneal burns: a quantitative comparison of acid and base. Acta Opbthalmol. 1984;62:542-548. 14. Roos A, Boron WF. Intracellular pH. Physiol Rev. 1981 ;61:296 434. 15. Bonanno JA. Regulation of corneal epithelial intracellular pH. Optorn Vis Sci. 1991;68:582-586. 16. Riley M, Winkler B. Strong Pasteur effect in rabbit corneal endothelium preserves fluid transport under anaerobic conditions. J Physiol, 1990;426:81-93. 17. Laing R, Chiba K, Tsubota K, Oak S. Metabolic and morphologic changes in the corneal endothelium. Invest Ophthalmol Vis Sci. 1992;33:3315-3324. 18. Candia OA, Reinach PS. Thermodynamic analysis of active sodium and potassium transport in the frog corneal epithelium. Am J Physiol. 1982;242:F690-F698. 19. Fatt I, Bieber MT, Pye SD. Steady state distribution of oxygen and carbon dioxide in the in vivo cornea of an eye covered by a gas-permeable contact lens. AmJ Optom. 1969;46:3-l4.
