July 1999
Volume 40, Issue 8
Free
Glaucoma  |   July 1999
Effects of Na-K-2Cl Cotransport Regulators on Outflow Facility in Calf and Human Eyes In Vitro
Author Affiliations
  • Lama A. Al-Aswad
    Ophthalmology and
  • Haiyan Gong
    Ophthalmology and
  • David Lee
    Ophthalmology and
  • Martha E. O’Donnell
    Human Physiology and
  • James D. Brandt
    Ophthalmology of the School of Medicine, University of California, Davis.
  • William J. Ryan
    Ophthalmology and
  • Alison Schroeder
    Ophthalmology and
  • Kristine A. Erickson
    Ophthalmology and
    Pharmacology of Boston University School of Medicine, Massachusetts;
    The New England College of Optometry, Boston, Massachusetts; and the Departments of
Investigative Ophthalmology & Visual Science July 1999, Vol.40, 1695-1701. doi:
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      Lama A. Al-Aswad, Haiyan Gong, David Lee, Martha E. O’Donnell, James D. Brandt, William J. Ryan, Alison Schroeder, Kristine A. Erickson; Effects of Na-K-2Cl Cotransport Regulators on Outflow Facility in Calf and Human Eyes In Vitro. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1695-1701.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Cultured human trabecular meshwork (TM) cells possess substantial Na-K-Cl activity, which is involved in the regulation of TM cell volume. The hypothesis in the present study was that drugs that affect the cotransporter might alter aqueous humor outflow facility (C) in the intact eye. The effects of agents and conditions known to modulate Na-K-Cl cotransport activity and/or TM cell volume on C in perfused anterior segments were investigated.

methods. Human and calf eyes were dissected and perfused, and C was determined according to standard published methods. Perfusates with modified osmolarity were used to cause alterations in TM cell volume. Cl-free perfusate and/or bumetanide (10−5 M) was used to inhibit Na-K-Cl cotransport activity, and vasopressin (10−7 M, 10−8 M) was used to stimulate cotransport activity.

results. In human eyes, hypo-osmotic perfusate decreased C 12%, whereas hyper-osmotic perfusate increased C 44%. These changes lasted approximately 30 minutes, after which C began to normalize. Inhibition of Na-K-Cl cotransport using Cl-free medium or bumetanide resulted in facility increases of 27% and 22%, respectively. There was an additive increase in C with bumetanide plus Cl-free media. Stimulating Na-K-Cl cotransport with 10−8 M and 10−7 M vasopressin resulted in 28% and 35% decreases in C, respectively. The results were similar in calf eyes: Cl-free medium or bumetanide resulted in 41% and 52% increases in C, whereas 10−8 M and 10−7 M vasopressin resulted in 14% and 19% decreases in C, respectively.

conclusions. Modulation of Na-K-Cl cotransport results in changes in C that may be mediated in part by cell volume changes.

In previous studies we have evaluated the effects of various vasoactive substances on outflow resistance of human and primate eyes in vitro. To date, we have identified several hormones that may play a regulatory role in aqueous outflow dynamics. 1 2 3 4 5 6 7 8 O’Donnell et al. 9 have demonstrated the presence of a highly active Na-K-Cl cotransport system in trabecular meshwork (TM) that functions to regulate intracellular volume of the TM cells. They also have shown that a number of the agents that we have found to alter outflow facility (C) in the human eye also modulate cotransporter activity and TM cell volume. This has led to the hypotheses that the Na-K-Cl cotransporter mediates changes in C by regulating TM intracellular volume. 
The Na-K-Cl cotransporter functions to transport Na, K, and Cl across the plasma membrane in an electroneutral 1:1:2 ratio. Characteristic features of the Na-K-Cl cotransporter are that it requires the presence of Na, K, and Cl to operate, with the exception that Rb can quantitatively substitute for K and that it is inhibited by “loop” diuretics such as bumetanide, benzmetanide, and furosemide. 10 11 12 13 14 15 The cotransporter can mediate both influx and efflux of Na, K, and Cl into and out of the cell. However, in TM cells, as in many other cells, the transporter mediates a net uptake of the ions into the cell (i.e., influx exceeds efflux). 9  
The Na-K-Cl cotransporter functions in regulation of intracellular volume for a variety of cell types. It functions to restore intracellular volume after hypertonicity-induced cell shrinkage, to maintain intracellular volume under isotonic conditions, and to mediate hormone-driven changes in intracellular volume. Thus, exposing TM cells to hypertonic media causes an immediate cell shrinkage as water exits the cell. If activity of the cotransporter is blocked by an inhibitor such as bumetanide, under these conditions the cells cannot reswell, indicating that Na-K-Cl cotransport activity is essential for this volume restoration, known as the regulatory volume increase (RVI). 10 11 12 13 14 15 Also, simply adding bumetanide under isotonic conditions causes the cells to shrink. This indicates that the cotransporter also functions to help maintain resting cell volume by mediating net uptake of Na, K, and Cl and thereby balancing net efflux occurring through other pathways, such as the NaK pump and the K and Cl channels. Finally, agents, such as vasopressin, that stimulate cotransport activity cause an increase in TM intracellular volume. In this regard, local hormones and other agents that may modify activity of the TM cell Na-K-Cl cotransporter are predicted to alter TM cell volume 9 and, in turn, to modify outflow of aqueous humor across the TM. 
The present study was conducted to evaluate the effects of agents known to modulate TM cell Na-K-Cl cotransport activity and/or TM cell volume on C of perfused anterior segments. 
Methods
Human and Calf Eyes and Perfusion Technique
Human and calf eyes were dissected and perfused according to standard published methods. 16 17 All data are expressed as mean ± SEM. A brief description of the method follows. 
Human eyes (n = 29; age 75 ± 1.87 years) with no history of ocular disease or surgery were obtained from the National Disease Research Interchange (Philadelphia, PA). The eyes were enucleated within 3 hours of the donors’ deaths and stored refrigerated in a humid saline environment until dissection. Within 12 hours of the donor’s death, the eyes were dissected at the eye bank, placed in optisol (Dexol; Chiron Ophthalmics, Irvine, CA) on ice, and shipped by overnight mail. The average time elapsed between donor death and initiation of baseline perfusion was 31.72 ± 1.93 hours. This protocol has been successful in ensuring that ocular tissue is viable at the time of perfusion. 16 17  
Neonatal calf eyes (n = 19; age, 3–5 days) were prepared for perfusion and perfused according to standard published techniques. 17 Briefly, the eyes were obtained from a local abattoir. They were placed on ice immediately after enucleation and delivered to the laboratory within 2 hours. Within 30 minutes of arrival in the laboratory, they were rinsed and dissected for perfusion. 
Before perfusion, human and calf eyes were rinsed in sterile Dulbecco’s modified Eagle’s medium (DMEM) containing 50 U/ml penicillin, 50 μg/ml streptomycin, and 5 μg/ml amphotericin B. 
The eyes were mounted on specialized perfusion chambers described previously 16 17 which were placed in an incubator at 37°C in a humid 5% CO2 environment. Perfusion was carried out at a constant pressure of 15 mm Hg, and outflow determinations were made under steady state conditions before and after experimental manipulation. 
Drugs and Chemicals
Dulbecco’s phosphate-buffered saline (DPBS; 0.88 mM calcium chloride · H2O, 0.49 mM magnesium chloride · 6H2O, 2.68 mM potassium chloride, 1.47 mM potassium phosphate monobasic [anhydrous], 136.9 mM sodium chloride, 0.81 mM sodium phosphate dibasic [anhydrous], 5.55 mM glucose, 289 ± 5% mOsm) containing 50 U/ml penicillin, 50 μg/ml streptomycin, and 5μ g/ml amphotericin B) was used as a buffer system for experiments investigating the effects of hyper-osmotic and hypo-osmotic media and Cl-free media on C. Hyper-osmotic perfusion medium (450 mOsmol) was prepared by adding 150 mM mannitol to DPBS. Hypo-osmotic perfusion medium (150 mOsmol) was made by omitting 75 mM NaCl from DPBS. Cl-free perfusion medium was prepared as a modification of DPBS by substituting every Cl salt with its gluconic acid equivalent. All media were iso-osmotic unless otherwise indicated. 
DMEM was used as the perfusion medium in experiments with bumetanide and vasopressin. A stock solution of bumetanide (10−2 M) was prepared in ethyl alcohol and then diluted into DMEM to a final concentration of 10−5 M. Similarly, vasopressin was dissolved in DMEM to make a stock solution that was finally diluted to working concentrations of 10−8 M and 10−7 M. 
All solutions and pharmacologic agents were obtained from Sigma, St. Louis, MO. 
Experimental Design and Statistical Analysis
Before testing the effects of the various media and pharmacologic agents on C, a baseline (in microliters per minute per millimeter mercury) determination of outflow facility (C0) was made after the tissue reached a steady state outflow. This determination consisted of the mean of determinations obtained every 15 minutes for 90 minutes. Thus, C0 consisted of the average of six facility determinations. After determination of C0, the perfusion chamber contents were exchanged with the test medium, a new steady state was obtained, and postdrug facility determinations (CD) were made at 15-minute intervals for either 30 minutes, in the case of the altered osmolarity experiments (the average of two measurements), or for 90 minutes, in the case of all other experiments (the average of six measurements). For experiments testing the effects of vasopressin, a sequential drug exchange protocol was used, involving 90-minute CD determinations after administration of 10−8 M vasopressin, followed by 90-minute CD determinations after administration of 10−7 M vasopressin. 
In human and calf eyes, the effects of anisosmotic media, Cl-free media, and bumetanide were evaluated by using a paired comparison of the ratios obtained in each eye of the average predrug and postdrug facilities (CD/C0). This method of data analysis normalizes the individual differences in baseline C and has been used extensively in outflow facility experiments. 1 2 3 4 5 6 7 8 16 17 18 19 20 21 22 23 Unlike C in calf and monkey eyes, facility of human eyes is well known to be stable for many hours. This is true of in vitro whole human eyes and cultured human anterior segments. 2 3 5 6 21 Therefore, in the case of human eyes, it is appropriate to compare postdrug and predrug facilities in a single eye. In contrast, calf eyes are well known to exhibit a progressive linear increase in C (washout) that occurs over the course of several hours of perfusion. Therefore, with calf eyes it is necessary to use paired control eyes to correct for the washout effect. 17 21 To correct for washout in these experiments, the CD/C0 ratio for the untreated eye was subtracted from the CD/C0 ratio of the treated eye, producing a washout-corrected ratio (CDC/C0). 
Statistical analysis consisted of comparisons between average predrug and postdrug facilities (CD/C0 or CDC/C0) using the paired two-tailed Student’s t-test. 
Tissue Fixation and Electron Microscopy
After perfusion with the experimental perfusate and determination of C, the human eyes were fixed by switching the perfusion fluid to modified Karnovsky’s fluid at the normal perfusion pressure of 15 mm Hg. The anterior segment of each eye was divided into four quadrants. From each quadrant, a series of radially oriented wedges were cut. Specimens were postfixed in 1% OsO4 and 1.5% potassium ferrocyanide in distilled water, dehydrated, and embedded in an Epon–Araldite mixture. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined with an electron microscope (model 300; Philips, Mahwah, NJ). 
Results
Perfusion of human ocular anterior segments with hypo-osmotic media, which are known to swell TM cells, resulted in a visibly larger cell size relative to an iso-osmotic control eye (Figs. 1 A, 1C). In contrast, perfusion with hyper-osmotic media resulted in an apparent marked decrease in cell size relative to the iso-osmotic perfused control eye (Figs. 1A 1B) . In all cases, cells lined the TM, the cells appeared to be viable, membranes were intact, and there was a normal chromatin pattern. This is typical of tissue specimens processed according to our stringent postmortem handling and time constraints, as previously described. 5 16 17  
Hypo-osmotic media resulted in a significantly decreased C, whereas perfusion with hyper-osmotic media, which shrinks the cells, caused an increase in C (Table 1) . Figure 2 depicts the results of a representative experiment: human anterior chamber segments were perfused with iso-osmotic medium to obtain a baseline value for C, followed by 90 minutes of perfusion with hypo-osmotic medium (150 mOsm), 90 minutes of perfusion with hyper-osmotic medium (450 mOsm), and then an additional, repeated treatment with hypo-osmotic medium (90 minutes) and hyper-osmotic medium (90 minutes). We found that perfusion of the anterior chamber with hypo-osmotic medium caused a decrease in the C ratio (CD/C0) that persisted for approximately 30 minutes, followed by a return to baseline C by approximately 45 minutes. A subsequent perfusion of the same anterior chamber with hyper-osmotic medium caused a significant elevation of C which was also transient, persisting for at least 30 minutes with a return to baseline C by approximately 45 minutes. Subsequent changes in media osmolarity showed that within a given eye, changes in osmolarity resulted in predictable and repeatable parallel changes in C (Fig. 2) . The mean effects of hypo-osmotic and hyper-osmotic media observed in several experiments are shown in Table 1 . In those experiments, an initial facility determination was made in iso-osmotic media followed by an exchange with either hyper-osmotic or hypo-osmotic media. Hypo-osmotic media significantly decreased C by an average of 17% ± 7% (P < 0.05), whereas hyper-osmotic media significantly increased C by 43% ± 12% (P < 0.01) in human eyes. 
To determine whether inhibition of Na-K-Cl cotransport activity affects C as predicted by our hypothesis, we examined C after perfusing human anterior segments with the Na-K-Cl cotransport inhibitor bumetanide (10−5 M) at a concentration known to inhibit cotransport activity maximally and to shrink cultured TM cells. 15 In the 15 experiments summarized in Table 1 we found that bumetanide increased C during the 90-minute postdrug period by a mean value of 23% ± 8% (P < 0.01). This effect persisted for the entire 90-minute post-bumetanide perfusion period (Fig. 3) . Similar results were obtained in perfused anterior segments of calf eyes, i.e., bumetanide caused a mean increase in C of 52% ± 15% (P < 0.05) as shown in Table 1
As another approach to examining the effect of inhibiting Na-K-Cl cotransport activity, we perfused human anterior segments with Cl-free media. Because the presence of all three transported ion species (Na, K, and Cl) is required for activity of the cotransporter, omitting any one of these ions from the extracellular medium prevents cotransport-mediated ion uptake and, thus, similar to bumetanide inhibition of the cotransporter, shrinks the cells. In addition to blocking cotransporter-mediated uptake of ions, Cl-free medium also promotes loss of Cl from the cells through Cl efflux pathways (Cl channels and KCl cotransport). Under the conditions of Cl-free media in the absence of bumetanide, the cotransporter can also contribute to loss of Cl in the cell, because it operates in reverse to cause an efflux of Na, K, and Cl from the cell. 
The results of our studies examining the effects of Cl-free media are shown as a representative experiment in Figure 4 and as mean values of several experiments in Table 1 . When human anterior chambers were perfused with Cl-free media, an immediate increase in C was observed. For the experiment shown in Figure 4 , an increase of 42% was observed at the first time point measured after starting the perfusion (15 minutes), lasting for at least 90 minutes before returning to baseline. Cl-free medium was found to increase C an average of 26% ± 8% (P < 0.01) in human eyes and 45% ± 16% (P < 0.02) in calf eyes (Table 1)
We also evaluated the effects of bumetanide and Cl-free medium in combination and found them to be additive. Perfusion of human anterior segments with Cl-free medium plus bumetanide, after an initial perfusion with bumetanide in Cl-containing medium, caused a further increase in C; from a 40% increase with bumetanide alone to an approximately 100% increase with bumetanide plus Cl-free medium in the two representative experiments depicted (Fig. 5)
The hormone vasopressin has been shown previously by O’Donnell et al. 15 to stimulate activity of TM cell Na-K-Cl cotransport. Thus, to examine the effects of increasing cotransporter activity on C, we perfused human anterior segments with vasopressin. Perfusion of the segments with 10−8 M vasopressin caused a marked decrease in C. As shown in the representative experiment of Figure 6a decrease averaging approximately 35% was observed, and it persisted for the duration of the perfusion. A subsequent perfusion with 10−7 M vasopressin did not cause a further decrease in C. Table 1 shows that mean decreases of 28% ± 6% (P < 0.02) and 35% ± 8% (P < 0.05) were observed for 10−8 M and 10−7 M vasopressin, respectively, in perfused human eyes. A similar result was seen in calf anterior segments perfused with vasopressin, although the effect was smaller, with 10−8 M and 10−7 M vasopressin causing 14% ± 13% (P < 0.001) and 19% ± 16% (P < 0.001) decreases in C, respectively. 
Discussion
The findings of the present study suggest that Na-K-Cl cotransport activity and intracellular volume influence C. Together with previous findings and parallel morphologic findings, our results support the hypothesis that the cotransporter, through regulating intracellular volume of the TM cells, modulates C. 
The results of this study are consistent with those of Gual et al. 24 who showed in an identical preparation that hypo-osmotic and hyper-osmotic media result in decreased and increased C, respectively, in bovine eyes. However, our results conflict with those of Gabelt et al. 22 who found that bumetanide did not change C in monkey eyes in vivo or in perfused human eyes in vitro. There are a number of possible reasons for the differing results. In the case of the monkey studies, it is possible that there are species differences. Alternatively, the presence of anesthesia, the washout effect, 23 or effects on other parameters of aqueous dynamics present in the living eye and absent in our system may have masked bumetanide’s effect on C. In the case of the human eye perfusions, it is likely that the conflicting results were caused by differences in experimental techniques. Gabelt et al. 22 used a constant-flow perfusion technique, rather than the constant-pressure technique that we use. In the constant-flow technique, pressure varies in response to changes in C or because of technical difficulties (e.g., pulsation of the syringe pump used to deliver the media to the eyes). Therefore, pressure spikes are not uncommon over the short term in this method. This can result in instability in or even damage to the outflow tissue. 
Further, the postmortem handling of tissues in Gabelt et al. was different from the methods used in our study. In that study, tissue was accepted undissected for experimentation up to 24 hours after donor death. Our requirements are considerably more stringent, simply because tissue autolysis occurs within hours of death, and if the eye tissue is not enucleated, dissected, and refrigerated soon after death, the tissue is no longer hormone responsive, even though it may look acceptable by light microscopy. 16 17 Tissue viability appears to have been a major problem in the Gabelt study, because more than half of the human eyes were deemed “unacceptable.” Finally, in that study only five experiments were performed at one dose of bumetanide. It is doubtful, given the inherent instability in the short term of the constant-flow technique, that statistical power was sufficient to uncover an effect of bumetanide. 
Anisosmotic media are well known to induce rapid changes in intracellular volume of a variety of cell types, 9 10 11 12 15 25 followed by activation of cell volume regulatory mechanisms. In nearly all cells, hyper-osmotic media cause an immediate shrinkage as water exits the cell, moving down its concentration gradient. This is true of TM cells, vascular endothelial cells, and a variety of other cells as well. 9 10 11 12 15 25 Cell shrinkage causes activation of Na-K-Cl cotransport or Na–H exchange, depending on the cell type, which leads to an increased net uptake of ions and, as water follows, a reswelling of the cell. This process, termed RVI, generally occurs over a 30- to 60-minute period after initial exposure to the anisosmotic medium. As volume is restored in the cells, the ion transporters mediating the RVI decrease in activity, returning to prestimulus levels. O’Donnell et al. 9 have shown that in cultured TM cells, it is the Na-K-Cl cotransporter that mediates the RVI after exposure to hyper-osmotic media. Hypo-osmotic media, in contrast, cause a rapid swelling of most cells, as water enters down its concentration gradient. Cell swelling immediately activates ion flux pathways that allow net efflux of ions from the cell. Water loss follows, and the cell is restored to its original volume. K and Cl channels and/or KCl cotransport, depending on the cell type, mediate this regulatory volume decrease. We do not yet know the pathways responsible for the regulatory volume decrease response in TM cells. 
Our finding that C is rapidly increased on exposure to hyper-osmotic media is consistent with the apparent cell shrinkage shown in Figure 1 and the rapid cell shrinkage of cultured TM cells caused by hyper-osmotic media. 9 The rapid decrease in C observed with hypo-osmotic media perfusion of anterior segments is similarly consistent with the rapid cell swelling observed in the experiment (Fig. 1) and in cultured TM 9 with hypo-osmotic media. Further, Rohen et al. 25 reported very similar results after perfusing monkey eyes with hyper-osmotic and hypo-osmotic media. Interestingly, they too reported that hypertonic media increases C, whereas hypotonic media decreases C. 25 The finding that the increased C observed with hyper-osmotic media is transient and reversible by 45 minutes is consistent with the time course of the RVI observed in cultured TM cells. 9  
If Na-K-Cl cotransport activity plays a role in maintaining and regulating TM C, it would be expected that inhibiting the transport activity would lead to a net sustained decrease in cell volume even in iso-osmotic conditions. O’Donnell et al. 9 have shown that the cotransport inhibitor bumetanide decreases the intracellular volume of cultured TM cells, as it does in a number of other cell types, including vascular endothelial cells. 9 10 11 12 15 25 Unlike the transient effects of hyper-osmotic media, bumetanide causes a cell shrinkage that is sustained up to at least 350 minutes. As would be predicted if TM cell volume is a determinant of C, we found that perfusing anterior chamber segments with bumetanide caused a sustained increase in C in both human and calf eyes. However, it is important to note that the cotransporter is electroneutral so that changes in its activity do not cause changes in membrane potential. In keeping with this, Wiederholt et al. 26 have recently reported that bumetanide does not alter the contractility of cultured TM cells. 
Perfusion of anterior chamber segments with Cl-free media, a maneuver that both inhibits cotransport activity and promotes reduction of cell volume through cotransport-independent pathways, was found to be another treatment that increased C. The finding that its effects were additive with those of bumetanide is consistent with a greater reduction in cell volume occurring than with bumetanide alone. 
A number of hormones are known to modulate TM C. The effects of some of these on cultured TM cell Na-K-Cl cotransport activity have been examined previously. 9 14 In those studies, norepinephrine acting through a β-adrenergic pathway, inhibited TM cotransport activity. In addition, elevation of cyclic adenosine monophosphate both inhibited cotransport activity and reduced TM cell volume. This is consistent with the well-known outflow-increasing effects ofβ -adrenergic agents. In contrast, the hormone vasopressin, acting through elevation of intracellular Ca and/or activation of protein kinase C, was shown to stimulate activity of the cotransporter. The finding in the present study that physiological doses of vasopressin caused a sustained decrease in C of both human and bovine anterior chamber segments suggests a role for hormone-driven TM cell volume changes in the modulation of C under iso-osmotic conditions. 
In summary, our studies provide evidence in support of the hypothesis that TM cell volume is a determinant of C and that the Na-K-Cl cotransporter plays a central role in this process, by regulating TM cell volume under isosmotic and anisosmotic conditions, and by mediating hormone-induced changes in cell volume. 
 
Figure 1.
 
The effect of hypo- and hyperosmotic media on the outflow pathway of human eyes. In human eyes perfused with PBS (A), the inner wall of Schlemm’s canal is intact and giant vacuoles can be seen. The inner wall, juxta canicular tissue, and the trabecular cells appear normal. When compared with human eyes perfused with PBS, eyes perfused with hyper-osmotic media (B) have cells with decreased cell volume in the inner wall of Schlemm’s canal, JCT, and beams; cells appear dark and shrunken. In human eyes perfused with hypo-osmotic media (C), the inner wall cells of Schlemm’s canal, JCT, and beams have increased cell diameter; nuclear regions of the cells appear lighter and swollen. SC: Schlemm’s canal. Original magnification, ×3730.
Figure 1.
 
The effect of hypo- and hyperosmotic media on the outflow pathway of human eyes. In human eyes perfused with PBS (A), the inner wall of Schlemm’s canal is intact and giant vacuoles can be seen. The inner wall, juxta canicular tissue, and the trabecular cells appear normal. When compared with human eyes perfused with PBS, eyes perfused with hyper-osmotic media (B) have cells with decreased cell volume in the inner wall of Schlemm’s canal, JCT, and beams; cells appear dark and shrunken. In human eyes perfused with hypo-osmotic media (C), the inner wall cells of Schlemm’s canal, JCT, and beams have increased cell diameter; nuclear regions of the cells appear lighter and swollen. SC: Schlemm’s canal. Original magnification, ×3730.
Table 1.
 
Table 1.
 
Perfusion of Ocular Anterior Segments with Drugs or Conditions That Inhibit or Stimulate the Na-K-Cl Cotransporter
Table 1.
 
Table 1.
 
Perfusion of Ocular Anterior Segments with Drugs or Conditions That Inhibit or Stimulate the Na-K-Cl Cotransporter
Condition n C0 CD CD/C0 Range P<
Human eyes
Bumetanide
10−5 M 15 0.22 ± 0.02 0.27 ± 0.03 1.23 ± 0.08 0.55–1.79 0.01
Cl-free 12 0.28 ± 0.04 0.35 ± 0.06 1.26 ± 0.08 0.78–1.76 0.01
Vasopressin
10−8 4 0.43 ± 0.12 0.31 ± 0.08 0.72 ± 0.06 0.58–0.86 0.02
10−7 4 0.43 ± 0.12 0.28 ± 0.08 0.65 ± 0.08 0.46–0.85 0.05
Hyper-osmotic 11 0.45 ± 0.15 0.64 ± 0.23 1.43 ± 0.12 0.98–2.46 0.01
Hypo-osmotic 10 0.31 ± 0.04 0.26 ± 0.04 0.83 ± 0.07 0.55–1.27 0.05
n C0 Sham CD/C0 Drug CD/C0 Difference in CD/C0* Range P <
Calf eyes
Bumetanide
10−5 M 5 0.71 ± 0.13 1.12 ± 0.08 1.64 ± 0.10 0.52 ± 0.15 0.26–1.11 0.05
Cl-free 8 1.16 ± 0.22 1.45 ± 0.08 1.90 ± 0.15 0.45 ± 0.16 −0.10–1.21 0.02
Vasopressin
10−8 8 1.22 ± 0.16 1.24 ± 0.08 1.10 ± 0.13 −0.14 ± 0.13 −0.44–0.63 0.001
10−7 7 0.93 ± 0.09 1.31 ± 0.10 1.18 ± 0.15 −0.19 ± 0.16 −0.52–0.58 0.001
Hyper-osmotic 5 1.09 ± 0.17 1.55 ± 0.21 1.33 ± 0.08 −0.22 ± 0.20 −0.91–0.23 0.01
Figure 2.
 
The effect of hypo- and hyper-osmotic media on outflow facility in human eyes. An eye from a 74-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS of 150 mOsmol, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS at 450 mOsmol, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 2.
 
The effect of hypo- and hyper-osmotic media on outflow facility in human eyes. An eye from a 74-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS of 150 mOsmol, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS at 450 mOsmol, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 3.
 
The effect of bumetanide followed by bumetanide plus Cl-free media on C in human eyes. Two eyes, one from a 58-year-old man (▪) and another from a 75-year-old (○) man, were perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS without Cl along with 10−5 M bumetanide, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for these representative experiments.
Figure 3.
 
The effect of bumetanide followed by bumetanide plus Cl-free media on C in human eyes. Two eyes, one from a 58-year-old man (▪) and another from a 75-year-old (○) man, were perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS without Cl along with 10−5 M bumetanide, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for these representative experiments.
Figure 4.
 
The effect of Cl-free media on C in human eyes. An eye from a 77-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged with DPBS without Cl, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 4.
 
The effect of Cl-free media on C in human eyes. An eye from a 77-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged with DPBS without Cl, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 5.
 
The effect of bumetanide on C is sustained. One eye from a 58-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged with DMEM containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 5.
 
The effect of bumetanide on C is sustained. One eye from a 58-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged with DMEM containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 6.
 
The effect of 10−8 and 10−7 M vasopressin on C in human eyes. One eye from a 75-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged, first with DMEM containing 10−8 M vasopressin, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DMEM containing 10−8 M vasopressin, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 6.
 
The effect of 10−8 and 10−7 M vasopressin on C in human eyes. One eye from a 75-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged, first with DMEM containing 10−8 M vasopressin, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DMEM containing 10−8 M vasopressin, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
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Figure 1.
 
The effect of hypo- and hyperosmotic media on the outflow pathway of human eyes. In human eyes perfused with PBS (A), the inner wall of Schlemm’s canal is intact and giant vacuoles can be seen. The inner wall, juxta canicular tissue, and the trabecular cells appear normal. When compared with human eyes perfused with PBS, eyes perfused with hyper-osmotic media (B) have cells with decreased cell volume in the inner wall of Schlemm’s canal, JCT, and beams; cells appear dark and shrunken. In human eyes perfused with hypo-osmotic media (C), the inner wall cells of Schlemm’s canal, JCT, and beams have increased cell diameter; nuclear regions of the cells appear lighter and swollen. SC: Schlemm’s canal. Original magnification, ×3730.
Figure 1.
 
The effect of hypo- and hyperosmotic media on the outflow pathway of human eyes. In human eyes perfused with PBS (A), the inner wall of Schlemm’s canal is intact and giant vacuoles can be seen. The inner wall, juxta canicular tissue, and the trabecular cells appear normal. When compared with human eyes perfused with PBS, eyes perfused with hyper-osmotic media (B) have cells with decreased cell volume in the inner wall of Schlemm’s canal, JCT, and beams; cells appear dark and shrunken. In human eyes perfused with hypo-osmotic media (C), the inner wall cells of Schlemm’s canal, JCT, and beams have increased cell diameter; nuclear regions of the cells appear lighter and swollen. SC: Schlemm’s canal. Original magnification, ×3730.
Figure 2.
 
The effect of hypo- and hyper-osmotic media on outflow facility in human eyes. An eye from a 74-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS of 150 mOsmol, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS at 450 mOsmol, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 2.
 
The effect of hypo- and hyper-osmotic media on outflow facility in human eyes. An eye from a 74-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS of 150 mOsmol, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS at 450 mOsmol, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 3.
 
The effect of bumetanide followed by bumetanide plus Cl-free media on C in human eyes. Two eyes, one from a 58-year-old man (▪) and another from a 75-year-old (○) man, were perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS without Cl along with 10−5 M bumetanide, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for these representative experiments.
Figure 3.
 
The effect of bumetanide followed by bumetanide plus Cl-free media on C in human eyes. Two eyes, one from a 58-year-old man (▪) and another from a 75-year-old (○) man, were perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged, first with DPBS containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DPBS without Cl along with 10−5 M bumetanide, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for these representative experiments.
Figure 4.
 
The effect of Cl-free media on C in human eyes. An eye from a 77-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged with DPBS without Cl, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 4.
 
The effect of Cl-free media on C in human eyes. An eye from a 77-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of iso-osmotic DPBS. The chamber contents were then exchanged with DPBS without Cl, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 5.
 
The effect of bumetanide on C is sustained. One eye from a 58-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged with DMEM containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 5.
 
The effect of bumetanide on C is sustained. One eye from a 58-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged with DMEM containing 10−5 M bumetanide, perfusion continued, and postdrug outflow facilities (CD) were calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 6.
 
The effect of 10−8 and 10−7 M vasopressin on C in human eyes. One eye from a 75-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged, first with DMEM containing 10−8 M vasopressin, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DMEM containing 10−8 M vasopressin, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Figure 6.
 
The effect of 10−8 and 10−7 M vasopressin on C in human eyes. One eye from a 75-year-old man was perfused at a constant pressure of 15 mm Hg at 37°C in a humid 5% CO2 environment, as described in the Methods section. The initial perfusion medium under which the baseline C (C0) was determined consisted of DMEM. The chamber contents were then exchanged, first with DMEM containing 10−8 M vasopressin, perfusion continued, and postdrug outflow facilities (CD) were calculated. A second exchange occurred, this time with DMEM containing 10−8 M vasopressin, perfusion continued, and CD was again calculated. Data show the ratio of CD/C0 over time for this representative experiment.
Table 1.
 
Table 1.
 
Perfusion of Ocular Anterior Segments with Drugs or Conditions That Inhibit or Stimulate the Na-K-Cl Cotransporter
Table 1.
 
Table 1.
 
Perfusion of Ocular Anterior Segments with Drugs or Conditions That Inhibit or Stimulate the Na-K-Cl Cotransporter
Condition n C0 CD CD/C0 Range P<
Human eyes
Bumetanide
10−5 M 15 0.22 ± 0.02 0.27 ± 0.03 1.23 ± 0.08 0.55–1.79 0.01
Cl-free 12 0.28 ± 0.04 0.35 ± 0.06 1.26 ± 0.08 0.78–1.76 0.01
Vasopressin
10−8 4 0.43 ± 0.12 0.31 ± 0.08 0.72 ± 0.06 0.58–0.86 0.02
10−7 4 0.43 ± 0.12 0.28 ± 0.08 0.65 ± 0.08 0.46–0.85 0.05
Hyper-osmotic 11 0.45 ± 0.15 0.64 ± 0.23 1.43 ± 0.12 0.98–2.46 0.01
Hypo-osmotic 10 0.31 ± 0.04 0.26 ± 0.04 0.83 ± 0.07 0.55–1.27 0.05
n C0 Sham CD/C0 Drug CD/C0 Difference in CD/C0* Range P <
Calf eyes
Bumetanide
10−5 M 5 0.71 ± 0.13 1.12 ± 0.08 1.64 ± 0.10 0.52 ± 0.15 0.26–1.11 0.05
Cl-free 8 1.16 ± 0.22 1.45 ± 0.08 1.90 ± 0.15 0.45 ± 0.16 −0.10–1.21 0.02
Vasopressin
10−8 8 1.22 ± 0.16 1.24 ± 0.08 1.10 ± 0.13 −0.14 ± 0.13 −0.44–0.63 0.001
10−7 7 0.93 ± 0.09 1.31 ± 0.10 1.18 ± 0.15 −0.19 ± 0.16 −0.52–0.58 0.001
Hyper-osmotic 5 1.09 ± 0.17 1.55 ± 0.21 1.33 ± 0.08 −0.22 ± 0.20 −0.91–0.23 0.01
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