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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Physiology, University of Otago Medical School, Dunedin, New Zealand; Departments of 2Physiology, 3Ophthalmology, and 4Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Submitted 30 January 2008 ; accepted in final form 22 August 2008
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ABSTRACT |
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regulatory volume decrease; adenosine receptor agonists; Schlemm's canal cells; juxtacanalicular cells; trabecular meshwork cells
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Fluid flow through the trabecular pathway is pressure dependent. The increase in outflow resistance associated with glaucoma leads to an increase in intraocular pressure (IOP), thereby increasing outflow to match inflow in the new steady state. The increased IOP is a major risk factor in glaucoma, a leading cause of irreversible blindness throughout the world (38). Recent work suggests that a parallel uveoscleral pathway through the ciliary muscle and exiting through the ocular venous system may be more significant than previously thought, particularly in young primates (18, 47). The uveoscleral pathway has been thought to be relatively pressure insensitive and appears highly species dependent.
In vivo, the time course of IOP does not follow the marked circadian rhythm of inflow (3, 27), suggesting that the resistance to outflow through the trabecular pathway is regulated. For example, the resistance to outflow significantly increases during nocturnal hours coincident with the circadian reduction in inflow, serving to partially stabilize IOP (43). The precise sensory and target sites of this regulation are not known but it is likely that regulation occurs somewhere in the anatomical region of the corneoscleral TM, JCT, and inner wall of Schlemm's canal (SCI) (15, 22). The very low absolute magnitude of the outflow resistance has suggested that outflow proceeds between the cells rather than through them (22). However, cells in the outflow pathway must play a role since swelling the cells increases resistance, and shrinking the cells reduces resistance in human, nonhuman primate, and calf eyes (2, 20, 40). Increase in volume of the TM cells has been suggested to increase resistance by restricting the adjacent space through which outflow can proceed (35). Aqueous humor outflow might also be limited by passage through pores within and between the inner-wall cells of Schlemm's canal (15, 21) and through the leaky tight junctions between these cells (39). In addition, changes in cell volume might affect resistance indirectly by releasing extracellular messengers (17) or matrix metalloproteinases (42). These issues have been addressed (22) by structural studies of the intact outflow pathway, with or without cationized ferritin tracer, and by studies of isolated TM or SC cells. However, there have been no studies of potential differential function of the various cell types in the intact outflow pathway. In the present work, we demonstrate the feasibility of measuring the elemental contents within the different cell types by electron probe X-ray microanalysis of the intact tissue, including the first functional analysis of JCT cells. We also report the results of hypotonic challenge, known to increase trabecular outflow resistance, and of exposure to selective A1 and A2A adenosine receptor (AR) agonists, known to exert opposite effects on IOP (4, 6, 12, 13, 46).
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METHODS |
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Corneoscleral tissue, either dissected from whole eyes or as corneoscleral rims, were placed endothelial side up and divided into two to three samples. With the use of a sapphire knife, remaining iris tissue was gently removed. The preparation was incised through the sclera just posterior and parallel to the scleral spur. A parallel incision was made anterior to Schlemm's canal at an angle of 45° with the surface, intersecting the posterior incision at the scleral surface. The two to three samples of original corneoscleral tissue, containing corneoscleral TM, JCT, and SC with little uveal TM (uTM), were further divided radially in halves, transferred to Petri dishes, and incubated under control or experimental conditions for 30 min. Under control conditions, the tissues were incubated in isotonic Dulbecco's phosphate-buffered saline (290 mosmol/kgH2O, PBS, Invitrogen, Carlsbad, CA). Under experimental conditions, drugs were included in the PBS or PBS was diluted with water to generate 33% (200 mosmol/kgH2O) or 50% hypotonic solutions (152 mosmol/kgH2O). After incubation, excess fluid was adsorbed from the tissues with filter paper. The tissues were quick-frozen in liquid nitrogen-cooled propane to prevent redistribution of ions and water and stored in liquid nitrogen until further processing for microanalysis.
Frozen tissue was fractured into blocks under a dissecting microscope (x7). Sections were cut 0.6–0.8 µm thick at –110 to –115°C, freeze-dried at 10–4 Pa (equivalent to 7.5 x 10–7 Torr), and transferred to a JEOL JSM 840 scanning electron microscope equipped with an energy-dispersive X-ray spectrometer for analysis.
Data acquisition and reduction. Cell identification was facilitated by reference to SC (Fig. 2). Cells at the luminal surface were identified as outer or inner wall SC cells (SCO and SCI, respectively). Cells just subjacent to the SCI cells were taken to be JCT cells (Fig. 3). Cells more distantly removed were defined as TM cells. Most of the TM cells analyzed were likely of corneoscleral origin. However, a small number of cells may have been uTM in view of their inner position in tissue section (Fig. 3).
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0.6 x 0.8 µm to 1.5 x 2.0 µm, depending on the cell geometry. A probe current of 140–200 pA was applied for 100 s at an accelerating voltage of 20 kV. As in past studies (8, 29–33), the area scanned was chosen to preclude contamination with extracellular Na+ and Cl– that might contribute to measurements conducted at intracellular sites closer to the plasma membrane. Thus part or all of the nucleus was included. The incident electrons ionize only a small fraction of the irradiated atoms. When an electron is knocked out of an inner shell, an electron from an outer atomic shell can take its place. Relaxation from a higher to a lower energy state releases an X-ray photon. X-rays were collected and analyzed with a Tracor Northern 30 mm2, energy-dispersive X-ray detector. The element is identified from the X-ray energy while the number of quanta collected at each characteristic energy permits quantification of its content. As previously described, signals were quantified by filtered least-squares fitting to a library of monoelemental peaks (9). The Na, Mg, Si, P, S, Cl, K, and Ca spectra in that library were obtained from microcrystals sprayed onto a Formvar film. In addition to producing element-characteristic X-rays, electron beam irradiation generates nonquantal white or continuous radiation (Bremsstrahlung), arising from electron deceleration by coulombic interaction with atomic nuclei. These white counts (w), providing an index of tissue mass (10), were obtained over the energy range 4.6–6.0 keV and corrected for contaminant contributions from the Al specimen holder and Ni grid. As noted previously (30), Na, K, and Cl signals were normalized as molar ratios to the P signal determined in the same scanned area. Phosphorus is particularly appropriate for normalization because its intracellular signal is constant, almost entirely reflecting covalently linked atoms (e.g., Ref. 10). The normalization to P has been validated elsewhere by the close linear relationship between the two largely intracellular elements K and P (Fig. 3, Ref. 8).
Water is not directly measured by electron probe X-ray microanalysis. However, the sum of the normalized contents of Na and K [(Na+K)/P] has proved to be a useful index of intracellular water content (1). We have also used the normalized anion gap, defined as (Na+K-Cl)/P, as an approximate index of changes in intracellular HCO3– content (33), although other unmeasured anions can also contribute to this parameter.
Statistics. Elemental microanalyses of the different cell types under control and experimental conditions are presented as means ± SE in the tables and figures. To ensure robustness of the analyses, means were included only if based on at least 10 microanalyses. Unless otherwise stated, the probability (P) of the null hypothesis was estimated by Student's t-test, defining significance as P < 0.05. One-way ANOVA using the Bonferroni test was used to test for the significance of differences among multiple means.
Drugs. All drugs were purchased from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Figure 3 presents a tissue section illustrating cells identified as: inner wall (SCI) and outer wall (SCO) cells from SC, a JCT cell, a corneoscleral TM cell, and an uTM cell. The loop of tissue displaced downward just to the left of the scleral spur at the bottom of the figure was also likely of uTM origin. However, as illustrated here, the uTM was often fractured so that identifying uTM was less certain than recognizing SCI and SCO cells, JCT cells, and corneoscleral TM cells.
Intracellular elemental contents under baseline and ouabain-treated conditions. Table 1 presents the elemental contents of all cells analyzed in the trabecular outflow pathway under control conditions. The high K+ and P contents and low Na+ and Cl– contents documented that the areas analyzed were indeed intracellular. The phosphorus content (normalized to white counts), Na/P, Cl/P, K/P, cell volume [monitored as (Na+K)/P], and unmeasured anion contents [(Na+K-Cl)/P] were comparable to the contents of ciliary epithelial cells we have recently analyzed (32). Incubation with 100 µM ouabain for 30 min to block the Na+-K+-activated ATPase increased the mean Na/P and decreased K/P by an order of magnitude (N = 122) (Table 1, Fig. 4). The two cell types studied most completely after ouabain incubation, and included in Table 1, were the TM cells (N = 74) and the SCI cells (N = 20). Ouabain increased Na/P to 1.730 ± 0.055 and reduced K/P to 0.111 ± 0.015 in TM cells and raised Na/P to 1.506 ± 0.095 and reduced K/P to 0.104 ± 0.019 in the SC cells. These ouabain-produced changes did not simply reflect an exchange of intracellular K+ for extracellular Na+. With loss of cell K+, the membrane is expected to depolarize. The depolarization should permit influx of Cl–, counterbalanced by Na+, thus producing the observed increases in Cl/P and volume, followed as (Na+K)/P (Table 1).
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As noted in the METHODS, we do not measure volume directly, but follow changes in cell volume with the sum of the normalized elemental Na and K contents [(Na+K)/P]. When we reduce the external osmolality, water enters the cells. We cannot detect this initial anisosmotic cell swelling with our index of cell volume. Initially, the elemental contents will be unchanged, even when the cell is swollen. What we do detect is the regulatory response to this cell swelling, the release of solute, largely K+ and Cl–.
Figure 6, A and B, illustrates the reductions in total Cl– content and volume, respectively, produced by incubating the intact trabecular outflow pathway in hypotonic solution for 30 min. Table 3 presents the corresponding changes in normalized elemental contents and volume based on microprobe analysis of 985 cells under isosmotic and 1,096 cells under hypotonic conditions. From analysis of the total set of cells, the anisosmotic swelling triggered a significant decrease (P < 0.001) from baseline values (Table 2) of Na+, Cl–, K+, and unmeasured anion contents, together with a decrease in cell volume, monitored as (Na+K)/P. Based on Tables 2 and 3, the mean fall in volume of all the cells was 13.1 ± 0.7% (P < 0.011), largely reflecting K+ release. Half the accompanying anion release reflected Cl–, reducing intracellular Cl– by 25%, and half by unmeasured anion [(Na+K-Cl)/P], likely reflecting reduced intracellular HCO3–.
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Effects of purinergic agonists on intracellular elemental contents. The results presented in the figures and tables have been based on more than 3,000 microanalyses of individual cells, and 570 cells were analyzed in studying the effects of two A1 agonists. Nevertheless, the cells lining the surface of SC contribute a relatively small fraction of the total population of cells in the trabecular outflow pathway. Thus the number of SCI cells available for microanalysis proved to be limited. The results of incubating tissues with two selective A1 AR agonists were similar. To enhance the robustness of the calculation, the microanalyses conducted following exposure to the A1 agonists adenosine amine congener (ADAC, N = 14 SCI cells) and (2S)-N6-[2-endo-norbornyl]adenosine (S-ENBA, N = 17 SCI cells) were combined (N = 31 SCI cells) in Table 4 and Fig. 6. A comparable number of SCI cells (N = 36) were analyzed in studying the effects of the single selective agonist (CGS-21680) used to activate A2A ARs (Table 4, Fig. 6).
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The SCI responded uniquely to the purinergic agonists. Like the JCT, TM, and SL, the SCI cells gained Cl–, K+, and volume following incubation with the A2A agonist (Table 4, Fig. 6). In contrast, however, the A1 agonists did not significantly alter the Cl– and K+ contents and volume of the SCI cells (Table 4, Fig. 6).
ANOVA of Cl– content and volume for each cell type under control and experimental conditions. Figure 6, A and B, summarizes the mean Cl– contents and volume, respectively, for TM, JCT, and SCI cells, and for the total set of outflow cells analyzed under control and experimental conditions. The cellular responses to the experimental conditions fell into three patterns.
The first pattern was displayed by JCT, TM, and EW cells. One-way ANOVA of the microanalyses indicated that hypotonicity, A1 agonists and the A2A agonist all significantly changed (P < 0.05) both cell Cl– contents and cell volume. The magnitudes of the increases in Cl/P and volume of the JCT and EW cells were not significantly different after incubation with A1 (Table 4) and A2A agonists (Table 4), but the A2A agonist produced a significantly larger increase in volume than in Cl– contents of the TM cells (Table 4). The second pattern was displayed by SCI cells, whose Cl– contents or volume were not significantly altered by A1 agonists, but were significantly altered by either A2A or hypotonicity. A third pattern was identified by one-way ANOVA of the uTM cells, whose Cl– contents and cell volume were significantly changed by hypotonicity, but not by the A1 or A2A agonists (Table 4).
Of particular interest, the adjoining cell layers of SCI and JCT cells responded differently to agonists of A1 and A2A receptors. The A1 agonists significantly increased the Cl– contents and cell volumes of the JCT cells but not of the SCI cells (Table 4). In contrast, the A2A agonist increased the Cl– contents and cell volumes of both cell types (Table 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Feasibility of electron probe X-ray microanalysis of the trabecular outflow pathway. The small size, complexity, and cellular heterogeneity of the trabecular outflow tract have hindered progress in studying the cell physiology of the intact tissue. The present results demonstrate the feasibility of identifying the component cell types even in unfixed, unstained sections of the outflow pathway (Figs. 2 and 3). Microprobe analysis of the component cells revealed high baseline elemental P and K+ contents and low Na+ and Cl– contents, characteristic of intracellular sites under baseline conditions (Tables 1 and 2). Furthermore, blocking Na+-K+-activated ATPase with ouabain produced the large changes in Na+ and K+ contents characteristically displayed by other cells (Table 1, Fig. 4). These results document the feasibility of functionally studying cells in the human trabecular outflow pathway with electron probe X-ray microanalysis.
RVD of cells in the intact trabecular outflow pathway. Anisosmotic swelling of isolated human TM cells has previously been found to initiate a RVD that could be inhibited by blocking K+ or Cl– channels (34). Hypotonicity has also been reported to activate K+ and Cl– channels of primary cultures of bovine TM cells (44). Srinivas et al. (45) found swelling-activated Cl– channels in bovine TM cells but, in contrast to Soto et al. (44), did not observe swelling-activated K+ channels or an RVD. Among other possibilities, this might have reflected different origins of the bovine TM cells used. Isolated, cultured bovine and human TM cells express functional heterogeneity (11, 48), and histochemical staining of human TM cells reveals heterogeneous expression of myosin (14).
As noted in the METHODS and RESULTS, we do not measure volume directly but follow changes in cell volume with the measured sum of the normalized elemental Na and K contents [(Na+K)/P]. This index does not detect the initial cell swelling necessarily produced by hypotonic challenge because the total ion content will not change acutely with swelling but does permit us to monitor changes in volume as ions and hence water are released from the cell. The present results demonstrate that hypotonic challenge of the intact human trabecular outflow tissue produces a secondary loss of Cl– content and cell volume, monitored indirectly as (Na+ + K+)/P, by all cell types. By definition, this swelling-activated loss of cell volume conforms to a regulatory volume decrease (26). The magnitudes of the regulatory solute release expressed by the different cell types did not differ significantly from one another (P > 0.3, one-way ANOVA). The mean percentage loss in cell volume displayed by all cells analyzed after hypotonic incubation (N = 1,096) was 13.1 ± 0.7%.
We have previously quantified the RVD of isolated human TM cells by direct volumetric measurements (34). The 24% hypotonicity applied to the isolated TM cells of the previous study would be expected to swell perfect osmometers by 31%. The swelling observed was 29 ± 3% (means ± SE, N = 3), very close to the predicted value. Thereafter, both adherent TM cells (studied by fluorometry) and TM cells in suspension (studied by electronic cell sizing) displayed an RVD, a progressive shrinkage in the sustained presence of the hypotonic solution. Adherent cells displayed a regulatory shrinkage of 8.3% after 25 min of hypotonic challenge. The TM cells in suspension displayed shrinkages of 13% after 30 min and 19% after 60 min.
The 33% and 50% hypotonicity applied in the present study would swell perfect osmometers by 45% and 91%, respectively. Based on the data of Tables 2 and 3 in the present study, the 30-min hypotonic incubation was associated with a mean fall in normalized (Na + K) content of all outflow cells of 13.1 ± 0.7%. The surprisingly good quantitative agreement with the previous measurements may well be fortuitous, given the differences in technique, tissue preparation, hypotonic challenge, and other experimental conditions. However, the magnitude of the hypotonically triggered solute release was clearly comparable to the RVD directly measured in isolated cells.
Effects of A1 and A2A AR agonists on the different cell types of the intact outflow pathway. In general, selective A1 and A2A AR agonists produced similar changes in the Na+, K+, and Cl+ contents and volumes of all cell types (Table 4). The changes in Cl– contents (Fig. 6A) and cell volume (Fig. 6B) were of particular interest in view of the suggestion that changes in cell volume might mediate regulation of fluid flow through the trabecular outflow pathway (35). Consistent with this possibility, the Na+/H+ antiport inhibitor dimethylamiloride both shrinks isolated human TM cells (34) and reduces intraocular pressure (5). As expected, cell volume directly correlated with Cl– content under control conditions (Fig. 5).
Averaging the microanalyses of all outflow cells, the mean increases in Cl– contents (±SE) produced by A1 and A2A agonists were 18 ± 2% and 16 ± 2%, respectively. The corresponding increases in volume were 5 ± 1% and 10 ± 1%, respectively. The larger increase in volume triggered by the A2A agonist reflected an increase in unmeasured anion, presumably bicarbonate, in addition to the Cl– uptake (Table 4). These purinergic effects on the total set of cells were shared by the JCT, TM, and EW cells (Table 4). However, two cell types responded differently. First, the uTM cells responded neither to A1 nor A2A agonists, consistent with prior reports that the TM cells are functionally (11, 48) and immunohistochemically (14) heterogeneous. Second, the A2A agonist did significantly increase the Cl– content and cell volume of the SCI cells, but the A1 agonists affected neither parameter. These results may be relevant to the observation that activation of A1 and A2A ARs exert opposite effects on intraocular pressure (4, 6, 12, 13, 46).
Activation of A1 ARs lowers, and of A2A ARs increases, intraocular pressure in rabbits (12, 13), mice (4, 6), and monkeys (46). At least in monkeys, the A1-mediated reduction in IOP arises entirely from lowered resistance to outflow of aqueous humor (46). The A2A-mediated increase in IOP likely reflects an opposite effect on outflow resistance, since it is ascribable to neither increased inflow of aqueous humor (46) nor to interruption of the blood-aqueous barrier at low agonist doses (12, 13). If changes in cell volume mediate AR-activated changes in outflow resistance, we might predict that A1 agonists would shrink the target cells and A2A agonists should swell them.
The only cell type that responded differentially to A1 and A2A agonists was the SCI cell. A2A caused cell swelling and A1 agonists tended to cause cell shrinkage but not significantly so (Table 4). In part, this is consonant with our electrophysiological findings that A1 agonists increase and A2A agonists decrease, largely K+ currents in pure populations of cultured SCI cells (25). Activation of K+ channels should hyperpolarize the SCI membranes, favoring release of intracellular Cl– and shrink the cells, while depolarization is expected to reduce expulsion of Cl– and swell the cells. This suggests that SCI cell volume may play a role in regulating outflow resistance, but the regulation is likely to involve one or more additional factors. The underlying mechanisms are unclear, but the interaction of SCI and JCT cells may be particularly important.
The JCT has long been known to tether SCI cells in place through cytoplasmic connections (24), thereby preventing the SCI from ballooning into the lumen and obstructing aqueous humor outflow. Tethering is also supported by matrix-matrix interaction between the JCT and basement membrane of the SCI cells (19). However, the close proximity of the two cell types permits the JCT to modify SCI function, beyond simply tethering the SCI in place. Experimental maneuvers that physically separate the JCT from the SCI cells do lead to ballooning of the SCI and can permit the inner SC wall to contact the outer wall. Nevertheless, even in the presence of luminal obstruction in some local regions (28), separation of JCT and SCI reduces the resistance to aqueous humor outflow (28, 36, 41). The effect of separating JCT from SCI has been interpreted in terms of the funneling hypothesis that relates outflow resistance largely to hydrodynamic constraints on aqueous flow through the JCT to exit through pores between and through the SCI cells (23). Separation of JCT from SCI may relieve these constraints, reducing outflow resistance. However, quantitative agreement with the funneling hypothesis has been incomplete (16). An alternative possibility is that the JCT cells function analogously to pericytes regulating flow through capillaries (e.g., Ref. 37). Here, swelling of JCT and SCI cells follows A2A AR stimulation (which is associated with increased outflow resistance), and swelling of JCT but not SCI cells follows A2A AR stimulation (which is associated with reduced outflow resistance). Changes in the relative cell volumes of these adjoining cell types might possibly lead to changes in mechanical coupling and SCI cell pore formation.
Contribution of electron probe X-ray microanalysis. The present results indicate that cells in the intact trabecular outflow pathway likely function differently from those cells studied in isolation. First, reports of isolated cells have not agreed whether swelling of TM cells activates K+ channels and an RVD. In the intact tissue, we have now found that all cells of the outflow pathway, including the TM, express an RVD. Second, in contrast to previous studies of isolated human TM cells derived from mixed populations (34) or from a single cloned line (25), we have found that selective A1 and A2A agonists increase cell volume of TM cells in the intact tissue. In addition, electron probe X-ray microanalysis has permitted the first functional study of JCT cells in the intact outflow pathway, revealing that they respond to AR agonists differently from the adjoining layer of SCI cells. The microprobe has thus provided an unusual opportunity to begin studying the cell physiology of the trabecular outflow path.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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