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The ins and outs of aqueous humour secretion
Experimental Eye Research 78 (2004) 625–631 www.elsevier.com/locate/yexer
Review
The ins and outs of aqueous humour secretion Mortimer M. Civana,*, Anthony D.C. Macknightb a
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA b Department of Physiology, University of Otago Medical School, Dunedin, New Zealand Received 14 July 2003; accepted in revised form 25 September 2003
Abstract The intraocular pressure (IOP) reflects a balance between inflow and outflow of aqueous humour. A major strategy in the medical treatment of glaucoma is to reduce inflow and thereby IOP. Understanding the mechanisms and regulation of inflow is thus of clear clinical relevance. Many mechanisms underlying inflow have been identified. The integration and regulation of these mechanisms is less clear. Aqueous humour is secreted across the ciliary epithelium by transferring solute, chiefly NaCl, from the stroma to the posterior chamber of the eye, with water passively following. The epithelium consists of two layers: the pigmented ciliary epithelial (PE) cells abutting the stroma, and the non-pigmented ciliary epithelial (NPE) cells facing the aqueous humour. Gap junctions link adjacent cells within and between these layers. Secretion proceeds in three steps: (1) uptake of NaCl from stroma to PE cells by electroneutral transporters, (2) passage of NaCl from PE to NPE cells through gap junctions, and (3) release of Naþ and Cl2 through Naþ, Kþ-activated ATPase and Cl2 channels, respectively. Most of our understanding of inflow mechanisms has been obtained by studying in vitro preparations at subcellular, cellular and tissue levels. A particularly productive approach has been the electron probe X-ray microanalysis (EPMA) of the elemental composition of excised ciliary epithelium. This technique permits analysis of adjacent cells within different regions of the ciliary epithelium. EPMA of rabbit preparations has supported the idea that paired activity of Naþ/Hþ and Cl2/HCO2 3 antiports can be the dominant mechanism underlying the first step in secretion, stromal NaCl uptake by PE cells. EPMA also indicates that Cl2 turnover is faster in the anterior than the posterior region of the epithelium. At the opposite epithelial surface, release of Naþ through Naþ,Kþ-activated ATPase of NPE cells is also greater anteriorly than posteriorly. The accompanying release of Cl2 through ion channels is enhanced by agonists of A3 adenosine receptors (ARs). The concepts that paired antiport activity is important in stromal NaCl uptake and that A3ARs modulate NaCl release into the aqueous humour were based on in vitro studies. The potential relevance of these conclusions to in vivo conditions has been tested by measurements of IOP in the living mouse. The results have confirmed the predictions that inhibitors of Naþ/Hþ antiports lower IOP, and that A3AR agonists and antagonists raise and lower IOP, respectively. q 2003 Elsevier Ltd. All rights reserved. Keywords: intraocular pressure; ciliary epithelial topology; electron microprobe analysis; sodium/hydrogen antiport; chloride/bicarbonate antiport; sodium–potassium-chloride symport; chloride channels; A3 adenosine receptors; aqueous humour reabsorption; functional topology
1. Introduction The intraocular pressure (IOP) is of physiologic and pathophysiologic importance. Constancy of the IOP maintains the curvature of the cornea and thereby maintains the refractive properties of the eye. On the other hand, IOP is commonly elevated in the glaucomas. Reducing IOP is the only medical approach known to delay the onset and slow the progression of blindness in this group of diseases * Corresponding author. Dr Mortimer M. Civan, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA. E-mail address: [email protected] (M.M. Civan). 0014-4835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2003.09.021
(Collaborative Normal-Tension Glaucoma Study Group, 1998a,b; The AGIS Investigators, 2000). The IOP reflects the balance between inflow and outflow of aqueous humour. Therefore, reducing inflow has been a major strategy in the medical therapy of the glaucomas. Aqueous humour is secreted by the ciliary epithelium, consisting of a pigmented ciliary epithelial (PE) cell layer facing the stroma and a non-pigmented ciliary epithelial (NPE) cell layer facing the posterior chamber of the eye. Adjoining cells both within and between the two layers are linked by gap junctions, so that the epithelium is a functional syncytium. As illustrated for the secretory pathway in the upper half of Fig. 1, much is known about the transport components underlying unidirectional
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Fig. 1. Model of aqueous humour formation. Mechanisms thought to underlie unidirectional secretion are presented in the upper pair of PE– NPE cells, and those possibly involved in unidirectional reabsorption are illustrated in the lower pair of PE– NPE cells (Civan, 1998; McLaughlin et al., 1998). Cell pairs located more anteriorly may be particularly important in secreting the aqueous humour, while those more posteriorly may possibly be involved in reabsorption (Ghosh et al., 1991; McLaughlin et al., 2001c, 2003).
secretion of solutes and water by the ciliary epithelium. In this consensus model of unidirectional secretion, aqueous humour is formed by transferring solute, principally NaCl, from stroma to posterior chamber, with water passively following the osmotic gradient. Unidirectional secretion comprises three sequential steps. First, NaCl is transferred from the stromal interstitium into the PE cells by one of the two sets of electroneutral transporters, paired Naþ/Hþ and þ þ 2 Cl2/HCO2 3 antiports or a Na – K –2Cl symport. Second, solute passes from the PE into the NPE cells through gap junctions. Finally, NPE cells release Naþ and Cl2 into the aqueous humour in large part through membrane Naþ,Kþactivated ATPase and Cl2 channels, respectively. With rare exception (Sears, 1984; Civan et al., 1996, 1997; McLaughlin et al., 1998), models of aqueous humour formation equate net inflow with unidirectional secretion across the ciliary epithelium. The tacit assumption is that unidirectional reabsorption by the ciliary epithelium from the aqueous humour back into the stroma is negligible. However, several studies have identified NPE- and PE-cell transporters that might underlie transfer of solute and water back from the posterior chamber to the stromal interstitium (lower reabsorptive pathway of Fig. 1). The final step in this putative reabsorption, release of Cl2 through PE-cell Cl2
channels, is expected to be enhanced by ATP-triggered generation of cAMP (Fleischhauer et al., 2001), known to act directly on the channels (Fleischhauer et al., 2001; Do et al., 2003).
2. Into the ciliary epithelium: NaCl uptake from the stroma As shown by Fig. 1, at least two electroneutral mechanisms have been thought to underlie NaCl uptake by the PE cells. Under certain conditions, uptake can be subserved by either paired Naþ/Hþ (NHE-1) and Cl2/ HCO2 3 (AE2) antiports (Wiederholt et al., 1991; Kaufman and Mittag, 1994; McLaughlin et al., 1998; Counillon et al., 2000; To et al., 2001) or a bumetanide-sensitive Naþ –Kþ – 2Cl2 symport (Wiederholt and Zadunaisky, 1986; Edelman et al., 1994; Crook et al., 2000; Do and To, 2000; Dunn et al., 2001; To et al., 2001). Electron probe X-ray microanalysis (EPMA) offers an unusual opportunity to assess the relative contributions of these NaCl uptake mechanisms (pp. 112 –125, Civan, 1983; Bowler et al., 1996). The strength of this technique is the capability of measuring Na, K and Cl content simultaneously within the same PE or NPE
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cell. Thus, we can measure the effect of blocking each transporter on the Cl content of the ciliary epithelial cells. In practice, rabbit iris-ciliary bodies are excised, divided into quarters and attached to Mylar frames with cyanoacrylate before incubation in beakers (Bowler et al., 1996) or chambers (McLaughlin et al., 2001a). Tissues are quickfrozen in liquid propane, cryosectioned and freeze-dried prior to analysis. Unstained PE and NPE cells can be readily identified (Bowler et al., 1996). An area clearly within the cell is irradiated with an electron beam, ionizing a small fraction of the atoms bombarded. Removal of an electron from an inner atomic shell permits an electron in an outer shell to take its place. Relaxation of the electron from the higher to the lower energy level produces a quantum of X-ray energy characteristic of the element. Quantification of the number of quanta emitted at each energy level allows measurement of the elemental content of the cell. These values are normalized to the phosphorus content of the sample (Bowler et al., 1996), since the great bulk of intracellular phosphorus is covalently linked and therefore unaffected by changes in transport activity. The absolute phosphorus content of NPE and PE cells is , 400 – 500 mmol kg21 dry weight (Bowler et al., 1996). Fig. 2 presents the results in classical bar graph form, providing means ^ S.E . Although convenient for presentation in this familiar form, data analyses were actually conducted with non-parametric statistical approaches without assuming normal distributions. As indicated in the figure, exposure to solutions containing CO2/HCO2 3 increased the Cl/P by , 0·06, corresponding to an increase in baseline Cl content of , 20 – 25% (Bowler et al., 1996; McLaughlin et al., 1998). Even with CO2/HCO2 3 present,
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reducing the rate of delivery of Hþ and HCO2 3 by blocking carbonic anhydrase with acetazoleamide lowered Cl/P (Fig. 2; McLaughlin et al., 1998). In the nominal absence of CO2/ HCO2 3 , blocking carbonic anhydrase should have no effect and indeed under those conditions, acetazoleamide did not affect Cl/P (Fig. 2; McLaughlin et al., 1998). Interestingly, the selective Naþ/Hþ antiport inhibitor dimethylamiloride (DMA) also reduced Cl/P of cells bathed with CO2/HCO2 3 (Fig. 2; McLaughlin et al., 2001b). These results, obtained by restoring CO2/HCO2 3 , blocking carbonic anhydrase activity and inhibiting Naþ/Hþ exchange demonstrated that paired Naþ/Hþ and Cl2/HCO2 3 antiport activity strongly modulates Cl2 uptake by ciliary epithelial cells. Blocking Naþ – Kþ – 2Cl2 co-transport with bumetanide also reduced the Cl content of the ciliary epithelial cells, but only in the nominal absence of CO2/HCO2 3 (Fig. 2). With CO2/HCO2 3 present, bumetanide unexpectedly increased the Cl content (Fig. 2). Evidently under the experimental conditions of these studies (McLaughlin et al., 1998), the paired antiports dominated the NaCl uptake step. However, in the presence of CO2/HCO2 3 , the intracellular NaCl was so elevated that the equilibrium thermodynamic driving force for solute uptake through the symport was exceeded. Now the symport was moving the ions out of the PE cell back to the stroma. This mechanism effectively prevents overload of the cells if the rate of uptake of NaCl from the stroma exceeds either the rate at which ions can pass through the gap junctions linking PE and NPE cells or the rate at which they can be lost from the NPE cells to the aqueous humour. Blocking Naþ – Kþ –2Cl2 co-transport with bumetanide under such conditions would reduce the rate of efflux of NaCl, thereby elevating both Na/P and Cl/P (Fig. 2).
Fig. 2. Dependence of intracellular Cl on electroneutral transporters of ciliary epithelial cells. Analyses based on results from McLaughlin et al. (1998, 2001c) in the form of bar graphs presenting means ^ S.E . NPE and PE cells were affected to similar extents in these experiments, so that data from them was combined.
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3. Out of the ciliary epithelium: NaCl release into the aqueous humour Chloride is the principal anion secreted by the ciliary epithelium, and under baseline conditions NPE cells display little Cl2-channel activity (Jacob and Civan, 1996). However, this channel activity can be up-regulated by several stimuli, including cell swelling (Yantorno et al., 1992) and inhibition of protein kinase C with staurosporine (Coca-Prados et al., 1995). Thus, it is likely that Cl2channel activity at the basolateral surface of the PE cells is a rate-limiting factor in the release of NaCl into the aqueous humour formation (secretory pathway, Fig. 1). Several lines of in vitro work have indicated that adenosine can activate NPE Cl2 channels by occupying A3 adenosine receptors (ARs): (1) mRNA is expressed both in immortalised human NPE cells and in rabbit ciliary epithelium (Mitchell et al., 1999); (2) A3AR agonists both activate Cl2 channels and stimulate shrinkage of immortalised human NPE cells (Mitchell et al., 1999; Carre´ et al., 2000); (3) A3AR antagonists prevent adenosine-activated shrinkage of the cultured human NPE cells (Mitchell et al., 1999); and (4) A3AR agonists increase short-circuit current across rabbit iris-ciliary body in the direction consistent with activation of basolateral NPE Cl2 channels (Carre´ et al., 1997; Mitchell et al., 1999). The physiologic source of the adenosine is thought to be the NPE cells themselves, which can be stimulated to release ATP in vitro by either cell swelling or the calcium ionophore ionomycin (Mitchell et al., 1998). The ATP can then be metabolised to adenosine by ecto-enzymes (Mitchell et al., 1998).
example, NPE cells of calves express higher levels of a1/a2/ a3/b1/b2 isoforms of Naþ,Kþ-activated ATPase in the anterior than in the posterior region of the ciliary epithelium (Coca-Prados and Sanchez-Torres, 1998), while PE cells express a constant relative concentration of a1/b1 throughout the ciliary epithelium. These observations have led to the thought that the anterior region is primarily responsible for secretion with the posterior region involved in net reabsorption back towards the stroma (Ghosh et al., 1991). This hypothesis has been tested by application of EPMA. Microprobe analyses have demonstrated that the K/Na content is higher in the posterior region than the anterior region of rabbit ciliary epithelium, consistent with a lower turnover of Naþ and Kþ posteriorly. Very recent analyses using ouabain as a probe have supported this conclusion (McLaughlin et al., 2003). In addition, three different experimental perturbations produced greater changes in
4. Topology of aqueous humour formation The foregoing information suggests that unidirectional secretion strongly depends on paired antiport activity in supporting the initial uptake of stromal NaCl and on open communication through gap junctions between the PE and NPE cells (Fig. 1). The final step, NPE-cell release of NaCl into the aqueous humour, is likely regulated by A3ARs. In parallel with the unidirectional secretion, NPE- and PE-cell transporters have been identified that could support unidirectional reabsorption, thereby reducing net secretion of aqueous humour. The final step in this putative reabsorption should be enhanced by cAMP-mediated activation of PE Cl2 channels (Fleischhauer et al., 2001; Do et al., 2003). The cartoons of Fig. 1 might suggest that the rate of formation of aqueous humour is constant throughout the ciliary epithelium. Actually, a number of lines of evidence have suggested that net ciliary epithelial secretion is highly region-dependent (Flu¨gel and Lu¨tjen-Drecoll, 1988; Flu¨gel et al., 1989; Eichhorn et al., 1990; Ghosh et al., 1990, 1991; Eichhorn and Lu¨tjen-Drecoll, 1993; Flu¨gel et al., 1993; Dunn et al., 2001; McLaughlin et al., 2001c, 2003). For
Fig. 3. Responses of mouse IOP to topically applied inhibitors of Naþ/Hþ antiporters (dimethylamiloride, DMA) and of Naþ – Kþ –2Cl2 antiports (bumetanide) (Avila et al., 2002a). (A) DMA (1 mM in a 10-ml droplet) reduced IOP. Water was then added as a control to verify the patency of the micropipette by osmotically raising IOP. (B) Bumetanide itself had no significant effect on mouse IOP at 1- or 10-mM droplet concentration. (Reproduced with the permission of the Association for Research in Vision and Ophthalmology, the copyright holder.)
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the Cl content of the anterior than of the posterior epithelium, consistent with a higher turnover rate of Cl2 anteriorly, as well. The three experimental manipulations were: removing CO2/HCO2 3 from the external solutions, inhibiting carbonic anhydrase with CO2/HCO2 3 present, and inhibiting Naþ –Kþ – 2Cl2 co-transport with bumetanide. Unidirectional secretion is presented anteriorly in the cartoon of Fig. 1 to emphasize that the rate of NaCl turnover is likely higher in the anterior than in the posterior region of the ciliary epithelium.
5. Intraocular pressure in the living mouse The model of aqueous humour formation represented in Fig. 1 is based upon in vitro information obtained from many laboratories. Its potential relevance to aqueous humour dynamics has been addressed by measurements of IOP in the living mouse. This species is particularly favourable as the trabecular meshwork and Schlemm’s canal are better developed in the eye of the mouse than in that of other commonly studied species such as the cow and rabbit (Tamm et al., 1999). The IOP of human and mouse eyes also respond similarly to the application of anisosmotic
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solutions and drugs which reduce inflow or enhance outflow of aqueous humour (Avila et al., 2001a). The increasing availability of transgenic mice makes this species especially important, despite the considerable challenge in measuring IOP of the very small mouse eye. Recently, it has become possible to measure mouse IOP reliably, continuously and precisely over periods as long as 45 min. The Servo-Null Micropipette System used (Avila et al., 2001a) is based on an approach developed in Rushmer’s laboratory some four decades ago (Wiederhielm et al., 1964). A 5-mm exploring micropipette filled with highly conducting solution is advanced into the anterior chamber. The IOP displaces the baseline solution with lowconducting aqueous humour, increasing the micropipette resistance. The counter-pressure needed to restore the position of the fluid column is equal to the IOP. This technique has been exhaustively validated (Avila et al., 2001a) and has been used to test whether Naþ/Hþ antiport activity and A3AR-activated Cl2 channels are indeed important in modulating IOP, as suggested by the results discussed above. On the basis of the electron microprobe results of Fig. 2, we would predict that blocking Naþ/Hþ antiport activity would lower inflow and IOP, whereas blocking
Fig. 4. Responses to topical application first of direct or indirect inhibitors of Naþ/Hþ antiporters, and then of bumetanide (Avila et al., 2002a): (A) 1 mM DMA followed by 1 mM bumetanide; (B) 1 mM BIIB723 followed and then 1 mM bumetanide; (C) 55·4 mM dorzolamide followed by 1 mM bumetanide, and (D) 1 mM EIPA and then 1 mM bumetanide. Bumetanide consistently and significantly reduced IOP after first blocking the Naþ/Hþ antiporters. (Reproduced with the permission of the Association for Research in Vision and Ophthalmology, the copyright holder.)
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Naþ – Kþ – 2Cl2 symport activity would be ineffective unless the antiports were inactive. Topical application of dimethylamiloride (DMA), an inhibitor of Naþ/Hþ antiport activity, indeed produced a prompt and substantial decrease in baseline mouse IOP (Fig. 3(A); Avila et al., 2002a). In contrast, topical application of bumentanide, an inhibitor of Naþ – Kþ –2Cl2 co-transport, had no effect (Fig. 3(B); Avila et al., 2002a). Bumetanide has also been reported to have no effect on aqueous humour dynamics or IOP in monkeys (Gabelt et al., 1997). Blocking Naþ/Hþ antiport activity with other agents, either directly (BIIB723, EIPA) or indirectly by reducing carbonic anhydrase activity (dorzolamide) all lowered IOP by themselves (Fig. 4). As predicted by the model of Fig. 1, bumetanide did produce a substantial lowering of IOP if added after first blocking the Naþ/Hþ antiport (Fig. 4). Under these conditions, the bumetanide was effective at a concentration which by itself had no effect (Fig. 3(B)). The Servo-Null Micropipette System has also been exploited to test whether A3ARs play a role in regulating IOP. Under baseline conditions, A3AR agonists increase, and A3AR antagonists decrease, mouse IOP (Avila et al., 2001b, 2002b), as predicted from the in vitro results. Furthermore, knockout of the A3ARs reduces mouse IOP (Avila et al., 2002b). Thus, inhibition of Naþ/Hþ counter-transport and blocking/knockout of A3ARs separately reduced mouse IOP, consistent with expectations.
6. Conclusions Aqueous humour is secreted by the ciliary epithelium in three steps. Paired Naþ/Hþ and Cl2/HCO2 3 antiports play a major role in transferring NaCl from the stroma into the PE cells. NaCl then passes through gap junctions to the NPE cells. The final step, release of NaCl into the aqueous humour is regulated by A3ARs. In parallel with this unidirectional secretion, transport mechanisms which might underlie unidirectional reabsorption have been identified. The final step in this putative reabsorption is regulated by cAMP. Increased reabsorption would reduce net aqueous humour secretion. Recent electron microprobe analyses have supported the concept that net transport across the ciliary epithelium is highly region-specific. The presence of both secretory and reabsorptive pathways allows for a much more precise control of the ciliary epithelial contribution to IOP. The possible physiologic importance of Naþ/Hþ antiports and A3ARs has been recently addressed. Consistent with predictions based on in vitro studies, topical application of antiport inhibitors and A3AR antagonists significantly and substantially lower IOP in the living mouse.
Acknowledgements Supported in part by research grants EY08343 (M.M.C.), EY013624 (M.M.C.) and core grant EY01583 from the National Institutes of Health, USA and by a Project Grant from the Health Research Council of New Zealand and Lottery Health and by research grants EY08343 and EY01583 (for core facilities) from the NIH, USA.
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McLaughlin, C.W., Macknight, A.D.C., Civan, M.M., 2001. Effects of ouabain on ciliary epithelial cell composition. In: Abstr. Intern. Union Physiol. Sci. #651. McLaughlin, C.W., Peart, D., Purves, R.D., Carre´, D.A., Macknight, A.D., Civan, M.M., 1998. Effects of HCO2 3 on cell composition of rabbit ciliary epithelium: a new model for aqueous humor secretion. Invest. Ophthalmol. Vis. Sci. 39, 1631–1641. McLaughlin, C.W., Peart, D., Purves, R.D., Carre´, D.A., PetersonYantorno, K., Mitchell, C.H., Macknight, A.D., Civan, M.M., 2001b. Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells. Am. J. Physiol. Cell Physiol. 281, C865–C875. McLaughlin, C.W., Zellhuber-McMillan, S., Macknight, A.D.C., Civan, M.M., 2003. Topography of ouabain-sensitive transport across ciliary epithelium revealed by electron microprobe analysis. Ophthalmic Res. 35, 37. McLaughlin, C.W., Zellhuber-McMillan, S., Peart, D., Purves, R.D., Macknight, A.D., Civan, M.M., 2001c. Regional differences in ciliary epithelial cell transport properties. J. Membr. Biol. 182, 213– 222. Mitchell, C.H., Carre´, D.A., McGlinn, A.M., Stone, R.A., Civan, M.M., 1998. A release mechanism for stored ATP in ocular ciliary epithelial cells. Proc. Nat. Acad. Sci. USA 95, 7174–7178. Mitchell, C.H., Peterson-Yantorno, K., Carre´, D.A., McGlinn, A.M., CocaPrados, M., Stone, R.A., Civan, M.M., 1999. A3 adenosine receptors regulate Cl2 channels of nonpigmented ciliary epithelial cells. Am. J. Physiol. 276, C659– C666. Sears, M.L., 1984. Autonomic nervous system: adrenergic agonists. In: Sears, M.L., (Ed.), Pharmacology of the Eye, Springer, New York, pp. 193–248. Tamm, E.R., Russell, P., Piatigorsky, J., 1999. Development and characterization of an immortal and differentiated murine trabecular meshwork cell line. Invest. Ophthalmol. Vis. Sci. 40, 1392–1403. The AGIS Investigators, 2000. The advanced glaucoma intervention study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. Am. J. Ophthalmol. 130, 429– 440. To, C.H., Do, C.W., Zamudio, A.C., Candia, O.A., 2001. Model of ionic transport for bovine ciliary epithelium: effects of acetazolamide and HCO. Am. J. Physiol. Cell Physiol. 280, C1521–C1530. Wiederhielm, C.A., Woodbury, J.W., Kirk, S., Rushmer, R.F., 1964. Pulsatile pressures in the microcirculation of a frog’s mesentery. Am. J. Physiol. 207, 173–176. Wiederholt, M., Helbig, H., Korbmacher, C., 1991. Ion transport across the ciliary epithelium: lessons from cultured cells and proposed role of the carbonic anhydrase. In: Botre´, F., Gross, G., Storey, B.T. (Eds.), Carbonic Anhydrase, VCH, New York, pp. 232 –244. Wiederholt, M., Zadunaisky, J.A., 1986. Membrane potentials and intracellular chloride activity in the ciliary body of the shark. Pflu¨gers Arch 407, S112–S115. Yantorno, R.E., Carre´, D.A., Coca-Prados, M., Krupin, T., Civan, M.M., 1992. Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling. Am. J. Physiol. 262, C501–C509.
Review
The ins and outs of aqueous humour secretion Mortimer M. Civana,*, Anthony D.C. Macknightb a
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA b Department of Physiology, University of Otago Medical School, Dunedin, New Zealand Received 14 July 2003; accepted in revised form 25 September 2003
Abstract The intraocular pressure (IOP) reflects a balance between inflow and outflow of aqueous humour. A major strategy in the medical treatment of glaucoma is to reduce inflow and thereby IOP. Understanding the mechanisms and regulation of inflow is thus of clear clinical relevance. Many mechanisms underlying inflow have been identified. The integration and regulation of these mechanisms is less clear. Aqueous humour is secreted across the ciliary epithelium by transferring solute, chiefly NaCl, from the stroma to the posterior chamber of the eye, with water passively following. The epithelium consists of two layers: the pigmented ciliary epithelial (PE) cells abutting the stroma, and the non-pigmented ciliary epithelial (NPE) cells facing the aqueous humour. Gap junctions link adjacent cells within and between these layers. Secretion proceeds in three steps: (1) uptake of NaCl from stroma to PE cells by electroneutral transporters, (2) passage of NaCl from PE to NPE cells through gap junctions, and (3) release of Naþ and Cl2 through Naþ, Kþ-activated ATPase and Cl2 channels, respectively. Most of our understanding of inflow mechanisms has been obtained by studying in vitro preparations at subcellular, cellular and tissue levels. A particularly productive approach has been the electron probe X-ray microanalysis (EPMA) of the elemental composition of excised ciliary epithelium. This technique permits analysis of adjacent cells within different regions of the ciliary epithelium. EPMA of rabbit preparations has supported the idea that paired activity of Naþ/Hþ and Cl2/HCO2 3 antiports can be the dominant mechanism underlying the first step in secretion, stromal NaCl uptake by PE cells. EPMA also indicates that Cl2 turnover is faster in the anterior than the posterior region of the epithelium. At the opposite epithelial surface, release of Naþ through Naþ,Kþ-activated ATPase of NPE cells is also greater anteriorly than posteriorly. The accompanying release of Cl2 through ion channels is enhanced by agonists of A3 adenosine receptors (ARs). The concepts that paired antiport activity is important in stromal NaCl uptake and that A3ARs modulate NaCl release into the aqueous humour were based on in vitro studies. The potential relevance of these conclusions to in vivo conditions has been tested by measurements of IOP in the living mouse. The results have confirmed the predictions that inhibitors of Naþ/Hþ antiports lower IOP, and that A3AR agonists and antagonists raise and lower IOP, respectively. q 2003 Elsevier Ltd. All rights reserved. Keywords: intraocular pressure; ciliary epithelial topology; electron microprobe analysis; sodium/hydrogen antiport; chloride/bicarbonate antiport; sodium–potassium-chloride symport; chloride channels; A3 adenosine receptors; aqueous humour reabsorption; functional topology
1. Introduction The intraocular pressure (IOP) is of physiologic and pathophysiologic importance. Constancy of the IOP maintains the curvature of the cornea and thereby maintains the refractive properties of the eye. On the other hand, IOP is commonly elevated in the glaucomas. Reducing IOP is the only medical approach known to delay the onset and slow the progression of blindness in this group of diseases * Corresponding author. Dr Mortimer M. Civan, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA. E-mail address: [email protected] (M.M. Civan). 0014-4835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2003.09.021
(Collaborative Normal-Tension Glaucoma Study Group, 1998a,b; The AGIS Investigators, 2000). The IOP reflects the balance between inflow and outflow of aqueous humour. Therefore, reducing inflow has been a major strategy in the medical therapy of the glaucomas. Aqueous humour is secreted by the ciliary epithelium, consisting of a pigmented ciliary epithelial (PE) cell layer facing the stroma and a non-pigmented ciliary epithelial (NPE) cell layer facing the posterior chamber of the eye. Adjoining cells both within and between the two layers are linked by gap junctions, so that the epithelium is a functional syncytium. As illustrated for the secretory pathway in the upper half of Fig. 1, much is known about the transport components underlying unidirectional
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Fig. 1. Model of aqueous humour formation. Mechanisms thought to underlie unidirectional secretion are presented in the upper pair of PE– NPE cells, and those possibly involved in unidirectional reabsorption are illustrated in the lower pair of PE– NPE cells (Civan, 1998; McLaughlin et al., 1998). Cell pairs located more anteriorly may be particularly important in secreting the aqueous humour, while those more posteriorly may possibly be involved in reabsorption (Ghosh et al., 1991; McLaughlin et al., 2001c, 2003).
secretion of solutes and water by the ciliary epithelium. In this consensus model of unidirectional secretion, aqueous humour is formed by transferring solute, principally NaCl, from stroma to posterior chamber, with water passively following the osmotic gradient. Unidirectional secretion comprises three sequential steps. First, NaCl is transferred from the stromal interstitium into the PE cells by one of the two sets of electroneutral transporters, paired Naþ/Hþ and þ þ 2 Cl2/HCO2 3 antiports or a Na – K –2Cl symport. Second, solute passes from the PE into the NPE cells through gap junctions. Finally, NPE cells release Naþ and Cl2 into the aqueous humour in large part through membrane Naþ,Kþactivated ATPase and Cl2 channels, respectively. With rare exception (Sears, 1984; Civan et al., 1996, 1997; McLaughlin et al., 1998), models of aqueous humour formation equate net inflow with unidirectional secretion across the ciliary epithelium. The tacit assumption is that unidirectional reabsorption by the ciliary epithelium from the aqueous humour back into the stroma is negligible. However, several studies have identified NPE- and PE-cell transporters that might underlie transfer of solute and water back from the posterior chamber to the stromal interstitium (lower reabsorptive pathway of Fig. 1). The final step in this putative reabsorption, release of Cl2 through PE-cell Cl2
channels, is expected to be enhanced by ATP-triggered generation of cAMP (Fleischhauer et al., 2001), known to act directly on the channels (Fleischhauer et al., 2001; Do et al., 2003).
2. Into the ciliary epithelium: NaCl uptake from the stroma As shown by Fig. 1, at least two electroneutral mechanisms have been thought to underlie NaCl uptake by the PE cells. Under certain conditions, uptake can be subserved by either paired Naþ/Hþ (NHE-1) and Cl2/ HCO2 3 (AE2) antiports (Wiederholt et al., 1991; Kaufman and Mittag, 1994; McLaughlin et al., 1998; Counillon et al., 2000; To et al., 2001) or a bumetanide-sensitive Naþ –Kþ – 2Cl2 symport (Wiederholt and Zadunaisky, 1986; Edelman et al., 1994; Crook et al., 2000; Do and To, 2000; Dunn et al., 2001; To et al., 2001). Electron probe X-ray microanalysis (EPMA) offers an unusual opportunity to assess the relative contributions of these NaCl uptake mechanisms (pp. 112 –125, Civan, 1983; Bowler et al., 1996). The strength of this technique is the capability of measuring Na, K and Cl content simultaneously within the same PE or NPE
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cell. Thus, we can measure the effect of blocking each transporter on the Cl content of the ciliary epithelial cells. In practice, rabbit iris-ciliary bodies are excised, divided into quarters and attached to Mylar frames with cyanoacrylate before incubation in beakers (Bowler et al., 1996) or chambers (McLaughlin et al., 2001a). Tissues are quickfrozen in liquid propane, cryosectioned and freeze-dried prior to analysis. Unstained PE and NPE cells can be readily identified (Bowler et al., 1996). An area clearly within the cell is irradiated with an electron beam, ionizing a small fraction of the atoms bombarded. Removal of an electron from an inner atomic shell permits an electron in an outer shell to take its place. Relaxation of the electron from the higher to the lower energy level produces a quantum of X-ray energy characteristic of the element. Quantification of the number of quanta emitted at each energy level allows measurement of the elemental content of the cell. These values are normalized to the phosphorus content of the sample (Bowler et al., 1996), since the great bulk of intracellular phosphorus is covalently linked and therefore unaffected by changes in transport activity. The absolute phosphorus content of NPE and PE cells is , 400 – 500 mmol kg21 dry weight (Bowler et al., 1996). Fig. 2 presents the results in classical bar graph form, providing means ^ S.E . Although convenient for presentation in this familiar form, data analyses were actually conducted with non-parametric statistical approaches without assuming normal distributions. As indicated in the figure, exposure to solutions containing CO2/HCO2 3 increased the Cl/P by , 0·06, corresponding to an increase in baseline Cl content of , 20 – 25% (Bowler et al., 1996; McLaughlin et al., 1998). Even with CO2/HCO2 3 present,
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reducing the rate of delivery of Hþ and HCO2 3 by blocking carbonic anhydrase with acetazoleamide lowered Cl/P (Fig. 2; McLaughlin et al., 1998). In the nominal absence of CO2/ HCO2 3 , blocking carbonic anhydrase should have no effect and indeed under those conditions, acetazoleamide did not affect Cl/P (Fig. 2; McLaughlin et al., 1998). Interestingly, the selective Naþ/Hþ antiport inhibitor dimethylamiloride (DMA) also reduced Cl/P of cells bathed with CO2/HCO2 3 (Fig. 2; McLaughlin et al., 2001b). These results, obtained by restoring CO2/HCO2 3 , blocking carbonic anhydrase activity and inhibiting Naþ/Hþ exchange demonstrated that paired Naþ/Hþ and Cl2/HCO2 3 antiport activity strongly modulates Cl2 uptake by ciliary epithelial cells. Blocking Naþ – Kþ – 2Cl2 co-transport with bumetanide also reduced the Cl content of the ciliary epithelial cells, but only in the nominal absence of CO2/HCO2 3 (Fig. 2). With CO2/HCO2 3 present, bumetanide unexpectedly increased the Cl content (Fig. 2). Evidently under the experimental conditions of these studies (McLaughlin et al., 1998), the paired antiports dominated the NaCl uptake step. However, in the presence of CO2/HCO2 3 , the intracellular NaCl was so elevated that the equilibrium thermodynamic driving force for solute uptake through the symport was exceeded. Now the symport was moving the ions out of the PE cell back to the stroma. This mechanism effectively prevents overload of the cells if the rate of uptake of NaCl from the stroma exceeds either the rate at which ions can pass through the gap junctions linking PE and NPE cells or the rate at which they can be lost from the NPE cells to the aqueous humour. Blocking Naþ – Kþ –2Cl2 co-transport with bumetanide under such conditions would reduce the rate of efflux of NaCl, thereby elevating both Na/P and Cl/P (Fig. 2).
Fig. 2. Dependence of intracellular Cl on electroneutral transporters of ciliary epithelial cells. Analyses based on results from McLaughlin et al. (1998, 2001c) in the form of bar graphs presenting means ^ S.E . NPE and PE cells were affected to similar extents in these experiments, so that data from them was combined.
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3. Out of the ciliary epithelium: NaCl release into the aqueous humour Chloride is the principal anion secreted by the ciliary epithelium, and under baseline conditions NPE cells display little Cl2-channel activity (Jacob and Civan, 1996). However, this channel activity can be up-regulated by several stimuli, including cell swelling (Yantorno et al., 1992) and inhibition of protein kinase C with staurosporine (Coca-Prados et al., 1995). Thus, it is likely that Cl2channel activity at the basolateral surface of the PE cells is a rate-limiting factor in the release of NaCl into the aqueous humour formation (secretory pathway, Fig. 1). Several lines of in vitro work have indicated that adenosine can activate NPE Cl2 channels by occupying A3 adenosine receptors (ARs): (1) mRNA is expressed both in immortalised human NPE cells and in rabbit ciliary epithelium (Mitchell et al., 1999); (2) A3AR agonists both activate Cl2 channels and stimulate shrinkage of immortalised human NPE cells (Mitchell et al., 1999; Carre´ et al., 2000); (3) A3AR antagonists prevent adenosine-activated shrinkage of the cultured human NPE cells (Mitchell et al., 1999); and (4) A3AR agonists increase short-circuit current across rabbit iris-ciliary body in the direction consistent with activation of basolateral NPE Cl2 channels (Carre´ et al., 1997; Mitchell et al., 1999). The physiologic source of the adenosine is thought to be the NPE cells themselves, which can be stimulated to release ATP in vitro by either cell swelling or the calcium ionophore ionomycin (Mitchell et al., 1998). The ATP can then be metabolised to adenosine by ecto-enzymes (Mitchell et al., 1998).
example, NPE cells of calves express higher levels of a1/a2/ a3/b1/b2 isoforms of Naþ,Kþ-activated ATPase in the anterior than in the posterior region of the ciliary epithelium (Coca-Prados and Sanchez-Torres, 1998), while PE cells express a constant relative concentration of a1/b1 throughout the ciliary epithelium. These observations have led to the thought that the anterior region is primarily responsible for secretion with the posterior region involved in net reabsorption back towards the stroma (Ghosh et al., 1991). This hypothesis has been tested by application of EPMA. Microprobe analyses have demonstrated that the K/Na content is higher in the posterior region than the anterior region of rabbit ciliary epithelium, consistent with a lower turnover of Naþ and Kþ posteriorly. Very recent analyses using ouabain as a probe have supported this conclusion (McLaughlin et al., 2003). In addition, three different experimental perturbations produced greater changes in
4. Topology of aqueous humour formation The foregoing information suggests that unidirectional secretion strongly depends on paired antiport activity in supporting the initial uptake of stromal NaCl and on open communication through gap junctions between the PE and NPE cells (Fig. 1). The final step, NPE-cell release of NaCl into the aqueous humour, is likely regulated by A3ARs. In parallel with the unidirectional secretion, NPE- and PE-cell transporters have been identified that could support unidirectional reabsorption, thereby reducing net secretion of aqueous humour. The final step in this putative reabsorption should be enhanced by cAMP-mediated activation of PE Cl2 channels (Fleischhauer et al., 2001; Do et al., 2003). The cartoons of Fig. 1 might suggest that the rate of formation of aqueous humour is constant throughout the ciliary epithelium. Actually, a number of lines of evidence have suggested that net ciliary epithelial secretion is highly region-dependent (Flu¨gel and Lu¨tjen-Drecoll, 1988; Flu¨gel et al., 1989; Eichhorn et al., 1990; Ghosh et al., 1990, 1991; Eichhorn and Lu¨tjen-Drecoll, 1993; Flu¨gel et al., 1993; Dunn et al., 2001; McLaughlin et al., 2001c, 2003). For
Fig. 3. Responses of mouse IOP to topically applied inhibitors of Naþ/Hþ antiporters (dimethylamiloride, DMA) and of Naþ – Kþ –2Cl2 antiports (bumetanide) (Avila et al., 2002a). (A) DMA (1 mM in a 10-ml droplet) reduced IOP. Water was then added as a control to verify the patency of the micropipette by osmotically raising IOP. (B) Bumetanide itself had no significant effect on mouse IOP at 1- or 10-mM droplet concentration. (Reproduced with the permission of the Association for Research in Vision and Ophthalmology, the copyright holder.)
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the Cl content of the anterior than of the posterior epithelium, consistent with a higher turnover rate of Cl2 anteriorly, as well. The three experimental manipulations were: removing CO2/HCO2 3 from the external solutions, inhibiting carbonic anhydrase with CO2/HCO2 3 present, and inhibiting Naþ –Kþ – 2Cl2 co-transport with bumetanide. Unidirectional secretion is presented anteriorly in the cartoon of Fig. 1 to emphasize that the rate of NaCl turnover is likely higher in the anterior than in the posterior region of the ciliary epithelium.
5. Intraocular pressure in the living mouse The model of aqueous humour formation represented in Fig. 1 is based upon in vitro information obtained from many laboratories. Its potential relevance to aqueous humour dynamics has been addressed by measurements of IOP in the living mouse. This species is particularly favourable as the trabecular meshwork and Schlemm’s canal are better developed in the eye of the mouse than in that of other commonly studied species such as the cow and rabbit (Tamm et al., 1999). The IOP of human and mouse eyes also respond similarly to the application of anisosmotic
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solutions and drugs which reduce inflow or enhance outflow of aqueous humour (Avila et al., 2001a). The increasing availability of transgenic mice makes this species especially important, despite the considerable challenge in measuring IOP of the very small mouse eye. Recently, it has become possible to measure mouse IOP reliably, continuously and precisely over periods as long as 45 min. The Servo-Null Micropipette System used (Avila et al., 2001a) is based on an approach developed in Rushmer’s laboratory some four decades ago (Wiederhielm et al., 1964). A 5-mm exploring micropipette filled with highly conducting solution is advanced into the anterior chamber. The IOP displaces the baseline solution with lowconducting aqueous humour, increasing the micropipette resistance. The counter-pressure needed to restore the position of the fluid column is equal to the IOP. This technique has been exhaustively validated (Avila et al., 2001a) and has been used to test whether Naþ/Hþ antiport activity and A3AR-activated Cl2 channels are indeed important in modulating IOP, as suggested by the results discussed above. On the basis of the electron microprobe results of Fig. 2, we would predict that blocking Naþ/Hþ antiport activity would lower inflow and IOP, whereas blocking
Fig. 4. Responses to topical application first of direct or indirect inhibitors of Naþ/Hþ antiporters, and then of bumetanide (Avila et al., 2002a): (A) 1 mM DMA followed by 1 mM bumetanide; (B) 1 mM BIIB723 followed and then 1 mM bumetanide; (C) 55·4 mM dorzolamide followed by 1 mM bumetanide, and (D) 1 mM EIPA and then 1 mM bumetanide. Bumetanide consistently and significantly reduced IOP after first blocking the Naþ/Hþ antiporters. (Reproduced with the permission of the Association for Research in Vision and Ophthalmology, the copyright holder.)
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Naþ – Kþ – 2Cl2 symport activity would be ineffective unless the antiports were inactive. Topical application of dimethylamiloride (DMA), an inhibitor of Naþ/Hþ antiport activity, indeed produced a prompt and substantial decrease in baseline mouse IOP (Fig. 3(A); Avila et al., 2002a). In contrast, topical application of bumentanide, an inhibitor of Naþ – Kþ –2Cl2 co-transport, had no effect (Fig. 3(B); Avila et al., 2002a). Bumetanide has also been reported to have no effect on aqueous humour dynamics or IOP in monkeys (Gabelt et al., 1997). Blocking Naþ/Hþ antiport activity with other agents, either directly (BIIB723, EIPA) or indirectly by reducing carbonic anhydrase activity (dorzolamide) all lowered IOP by themselves (Fig. 4). As predicted by the model of Fig. 1, bumetanide did produce a substantial lowering of IOP if added after first blocking the Naþ/Hþ antiport (Fig. 4). Under these conditions, the bumetanide was effective at a concentration which by itself had no effect (Fig. 3(B)). The Servo-Null Micropipette System has also been exploited to test whether A3ARs play a role in regulating IOP. Under baseline conditions, A3AR agonists increase, and A3AR antagonists decrease, mouse IOP (Avila et al., 2001b, 2002b), as predicted from the in vitro results. Furthermore, knockout of the A3ARs reduces mouse IOP (Avila et al., 2002b). Thus, inhibition of Naþ/Hþ counter-transport and blocking/knockout of A3ARs separately reduced mouse IOP, consistent with expectations.
6. Conclusions Aqueous humour is secreted by the ciliary epithelium in three steps. Paired Naþ/Hþ and Cl2/HCO2 3 antiports play a major role in transferring NaCl from the stroma into the PE cells. NaCl then passes through gap junctions to the NPE cells. The final step, release of NaCl into the aqueous humour is regulated by A3ARs. In parallel with this unidirectional secretion, transport mechanisms which might underlie unidirectional reabsorption have been identified. The final step in this putative reabsorption is regulated by cAMP. Increased reabsorption would reduce net aqueous humour secretion. Recent electron microprobe analyses have supported the concept that net transport across the ciliary epithelium is highly region-specific. The presence of both secretory and reabsorptive pathways allows for a much more precise control of the ciliary epithelial contribution to IOP. The possible physiologic importance of Naþ/Hþ antiports and A3ARs has been recently addressed. Consistent with predictions based on in vitro studies, topical application of antiport inhibitors and A3AR antagonists significantly and substantially lower IOP in the living mouse.
Acknowledgements Supported in part by research grants EY08343 (M.M.C.), EY013624 (M.M.C.) and core grant EY01583 from the National Institutes of Health, USA and by a Project Grant from the Health Research Council of New Zealand and Lottery Health and by research grants EY08343 and EY01583 (for core facilities) from the NIH, USA.
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