| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
From the Departments of 1 Ophthalmology and 2 Pharmacology of Boston University School of Medicine, Massachusetts; 3 The New England College of Optometry, Boston, Massachusetts; and the Departments of 4 Human Physiology and 5 Ophthalmology of the School of Medicine, University of California, Davis.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
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.
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 volume9 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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 previously16 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 5
µg/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 |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
|
|
|
|
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) .
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
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 TM9 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 ß-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 of ß-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.
|
Footnotes |
|---|
Supported in part by The Glaucoma Research Foundation, National Eye Institute Grant EY07321, The Massachusetts Lions Eye Research Fund, and Research to Prevent Blindness.
Submitted for publication November 9, 1998; revised February 22, 1999; accepted March 10, 1999.
Proprietary interest category: N.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. M. Dismuke, N. A. Sharif, and D. Z. Ellis Human Trabecular Meshwork Cell Volume Decrease by NO-Independent Soluble Guanylate Cyclase Activators YC-1 and BAY-58-2667 Involves the BKCa Ion Channel Invest. Ophthalmol. Vis. Sci., July 1, 2009; 50(7): 3353 - 3359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. W. McLaughlin, M. O. Karl, S. Zellhuber-McMillan, Z. Wang, C. W. Do, C. T. Leung, A. Li, R. A. Stone, A. D. C. Macknight, and M. M. Civan Electron probe X-ray microanalysis of intact pathway for human aqueous humor outflow Am J Physiol Cell Physiol, November 1, 2008; 295(5): C1083 - C1091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. M. Dismuke, C. C. Mbadugha, and D. Z. Ellis NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1378 - C1386. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Soto, J. Pintor, A. Peral, A. Gual, and X. Gasull Effects of Dinucleoside Polyphosphates on Trabecular Meshwork Cells and Aqueous Humor Outflow Facility J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1042 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. O. Karl, J. C. Fleischhauer, W. D. Stamer, K. Peterson-Yantorno, C. H. Mitchell, R. A. Stone, and M. M. Civan Differential P1-purinergic modulation of human Schlemm's canal inner-wall cells Am J Physiol Cell Physiol, April 1, 2005; 288(4): C784 - C794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Soto, N. Comes, E. Ferrer, M. Morales, A. Escalada, J. Pales, C. Solsona, A. Gual, and X. Gasull Modulation of Aqueous Humor Outflow by Ionic Mechanisms Involved in Trabecular Meshwork Cell Volume Regulation Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3650 - 3661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Gasull, E. Ferrer, A. Llobet, A. Castellano, J. M. Nicolas, J. Pales, and A. Gual Cell Membrane Stretch Modulates the High-Conductance Ca2+-Activated K+ Channel in Bovine Trabecular Meshwork Cells Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 706 - 714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Cohen, P. P. Lee, L. W. Herndon, P. Challa, O. Overbury, and R. R. Allingham Using the Arteriolar Pressure Attenuation Index to Predict Ocular Hypertension Progression to Open-angle Glaucoma Arch Ophthalmol, January 1, 2003; 121(1): 33 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Mitchell, J. C. Fleischhauer, W. D. Stamer, K. Peterson-Yantorno, and M. M. Civan Human trabecular meshwork cell volume regulation Am J Physiol Cell Physiol, July 1, 2002; 283(1): C315 - C326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. D. Stamer, K. Peppel, M. E. O'Donnell, B. C. Roberts, F. Wu, and D. L. Epstein Expression of Aquaporin-1 in Human Trabecular Meshwork Cells: Role in Resting Cell Volume Invest. Ophthalmol. Vis. Sci., July 1, 2001; 42(8): 1803 - 1811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Erickson and A. Schroeder Direct Effects of Muscarinic Agents on the Outflow Pathways in Human Eyes Invest. Ophthalmol. Vis. Sci., June 1, 2000; 41(7): 1743 - 1748. [Abstract] [Full Text]
|
|
|
|
|
|
B. Tian, B. Geiger, D. L. Epstein, and P. L. Kaufman Cytoskeletal Involvement in the Regulation of Aqueous Humor Outflow Invest. Ophthalmol. Vis. Sci., March 1, 2000; 41(3): 619 - 623. [Full Text]
|
|
Read all eLetters
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 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.
