Preface

inflow are provided in new chapters on the circulatory regulation and topography of inflow and on the potential coupling of inflow and outflow. Outflow and glaucoma are considered in greater breadth and depth, including new chapters on the pathogenesis of retinal ganglion cell death, on functional genomics, on clues to the molecular bases of glaucoma, and on innovative strategies for controlling intraocular pressure and for neuroprotection. Given the importance of whole animal studies and the conundra frequently arising from interpreting results obtained with diVerent species, the original single chapter has been expanded to two, with separate consideration of nonhuman whole animal models and of clinical studies.

In broadening the perspective, the number of authors contributing chapters has also increased. This necessarily leads to some overlap in subject material. I regard this overlap as positive in providing both emphasis of new, important concepts and in expressing a spectrum of views on those new concepts that are as yet incompletely accepted.

In addition to presenting new concepts, the second edition expands discussion of measurement techniques in isolated tissues, in nonhuman animals and in humans. These techniques include electron probe X ray microanalysis of in vitro tissues, measurements of the circulation, inflow and outflow of nonhuman and human subjects, and the techniques of functional genomics.

I express my appreciation to the contributors, both to the first edition and to this second edition of the book. I am also grateful to the reviewers who oVered constructive suggestions of the individual chapters: Drs. Nicholas A. Delamere, Tejvir S. Khurana, JeVrey W. Kiel, Michael H. Koval, Rajkumar V. Patil, W. Daniel Stamer, Richard A. Stone, and Chi ho To. Each of the authors has published significant contributions in journals. It is my hope that this book has succeeded in placing these contributions in a broader perspective, providing insight into seminal developments and future possibilities of addressing aqueous humor dynamics and glaucoma.

xviii Previous Volumes in Series

Volume 32 Membrane Fusion in Fertilization, Cellular Transport, and Viral Infection (1988)

Edited by Nejat Du¨zgu¨nes and Felix Bronner

Vol ume 33Mole cular Biology of Ionic Chann els* (1988) Edited by William S. Agnew, Toni Claudio, and Frederick J. Sigworth

Vol ume 34Cellul ar and Molecula r Biology of Sodium Tr ansport * (19 Edited by Stanley G. Schultz

Volume 35 Mechanisms of Leukocyte Activation (1990)

Edited by Sergio Grinstein and Ori D. Rotstein

Vol ume 36Pro tein–Mem brane Interactio ns* (1990)

Edited by Toni Claudio

Volume 37 Channels and Noise in Epithelial Tissues (1990)

Edited by Sandy I. Helman and Willy Van Driessche

Current Topics in Membranes

Vol ume 38Order ing the Mem brane Cyt oskele ton Tril ayer* (1991) Edited by Mark S. Mooseker and Jon S. Morrow

Volume 39 Developmental Biology of Membrane Transport Systems (1991)

Edited by Dale J. Benos

Volume 40 Cell Lipids (1994)

Edited by Dick Hoekstra

Vol ume 41Cell Biology and Mem brane Transpo rt Process es* (1994) Edited by Michael Caplan

Volume 42 Chloride Channels (1994)

Edited by William B. Guggino

Volume 43 Membrane Protein–Cytoskeleton Interactions (1996) Edited by W. James Nelson

Volume 44 Lipid Polymorphism and Membrane Properties (1997) Edited by Richard Epand

Volume 45 The Eye’s Aqueous Humor: From Secretion to Glaucoma (1998)

Edited by Mortimer M. Civan

Volume 46 Potassium Ion Channels: Molecular Structure, Function, and Diseases (1999)

Edited by Yoshihisa Kurachi, Lily Yeh Jan, and Michel Lazdunski

Volume 47 AmilorideSensitive Sodium Channels: Physiology and Functional Diversity (1999)

Edited by Dale J. Benos

Volume 48 Membrane Permeability: 100 Years since Ernest Overton (1999)

Edited by David W. Deamer, Arnost Kleinzeller, and Douglas M. Fambrough

Volume 49 Gap Junctions: Molecular Basis of Cell Communication in Health and Disease

Edited by Camillo Peracchia

Volume 50 Gastrointestinal Transport: Molecular Physiology Edited by Kim E. Barrett and Mark Donowitz

Volume 51 Aquaporins

Edited by Stefan Hohmann, Søren Nielsen and Peter Agre

Volume 52 Peptide–Lipid Interactions

Edited by Sidney A. Simon and Thomas J. McIntosh

Volume 53 CalciumActivated Chloride Channels

Edited by Catherine Mary Fuller

Volume 54 Extracellular Nucleotides and Nucleosides: Release, Receptors, and Physiological and Pathophysiological Effects Edited by Erik M. Schwiebert

Volume 55 Chemokines, Chemokine Receptors, and Disease Edited by Lisa M. Schwiebert

Volume 56 Basement Membrances: Cell and Molecular Biology Edited by Nicholas A. Kefalides and Jacques P. Borel

Volume 57 The Nociceptive Membrane

Edited by Uhtaek Oh

Volume 58 Mechanosensitive Ion Channels, Part A

Edited by Owen P. Hamill

Volume 59 Mechanosensitive Ion Channels, Part B

Edited by Owen P. Hamill

Volume 60 Computational Modelling of Membrane Bilayers Edited by Scott E. Feller

Volume 61 Free Radical Effects on Membranes

Edited by Sadis Matalon

I. OVERVIEW

In large part, this volume focuses on the aqueous humor, its inflow from the blood and its outflow from the eye into the venous circulation. This chapter addresses the first step in establishing that flow, the secretion of the aqueous humor by the ciliary epithelium. The major aims are to present the underlying transport components and regulatory elements of that secretion. The chapter will also introduce relatively recent changes in our thinking concerning the regulatory role of the circulation, functional topography and species variation in forming the aqueous humor. The latter issues will be addressed in depth in subsequent chapters

II. INTRODUCTION

A. Function of Aqueous Humor

One major function of aqueous humor inflow is to maintain inflation of the globe, stabilizing its optical properties. For this purpose, it might be expected that the intraocular pressure (IOP) of the eye would be relatively constant about the observed median of 16–17 mm Hg (Brubaker, 1998). Early reports of a circadian rhythm of IOP proved inconsistent (Liu, 1998; Asejczyk-Widlicka and Pierscionek, 2007). Furthermore, the variations in IOP of a few mm Hg observed during the day in individuals do not detectably alter image quality, presumably because of unidentified compensating mechanisms (AsejczykWidlicka and Pierscionek, 2007). A second major function of aqueous humor is to deliver oxygen and nutrients and to remove metabolic waste products from the avascular anterior segment consisting of the lens, cornea, and trabecular meshwork. Other functions ascribed to aqueous humor inflow have been less clearly defined (Krupin and Civan, 1996), and include the delivery of antioxidants, such as ascorbate, and participation in local immune responses. The ciliary epithelium concentrates ascorbate in the aqueous humor 40 fold over the plasma concentration (Krupin and Civan, 1996). In so doing, the intracellular ascorbate concentration of the ciliary epithelium likely increases to millimolar levels (Helbig et al., 1989b) through a Naþ ascorbate cotransporter (Socci and Delamere, 1988; Helbig et al., 1989b). This is comparable to the levels of ascorbate in the cerebrospinal fluid and brain cells (Rice, 2000). Recently, evidence has been reported that ascorbate may be a regulator of ion channel activity, and not simply a scavenger of reactive oxygen species (ROS) (Nelson et al., 2007). Ascorbate concentrations in the extracellular fluids of rat brain cycle during the day and can be correlated with total motor activity

(Fillenz and O’Neill, 1986). However, this ascorbate cycling in the brain is diurnal in being reversed by inverting the light–dark cycle, and cannot therefore be causally related to the circadian rhythm of aqueous humor inflow.

B. Inflow and Outflow Pathways

The aqueous humor is secreted by the ciliary epithelium into the posterior chamber bounded by the vitreous humor and lens posteriorly, and the iris and pupil anteriorly. The bulk of the fluid flows through the pupil into the anterior chamber, and finally exits at the angle formed by the iris and cornea. Most of the primate aqueous humor has long been considered to leave the anterior chamber through a ‘‘conventional’’ trabecular pathway (Bill and Phillips, 1971), consisting of the trabecular meshwork, juxtacanalicular tissue, Schlemm’s canal, collector channels, and venous outflow in series. More recent work has raised the possibility that a substantial fraction of the aqueous humor may exit through a complex, parallel uveoscleral outflow system. These outflow pathways are considered in depth in Chapters 6 (Freddo and Johnson, 2008), 7 (Toris, 2008), and 8 (Toris and Camras, 2008).

In contrast to IOP, the rate of inflow of aqueous humor undergoes an unequivocal and striking circadian rhythm. From 8 am to 12 pm, inflow in the normal young human reaches 3 ml/min, but falls by some 60% to1.3 ml/min from 12 to 6 am (Brubaker, 1998). Although the basis for this circadian rhythm is unclear (Toris and Camras, 2008), the magnitude of the decline is greater than that achievable by currently available drugs.

The rate of aqueous humor secretion can be altered by second messengers and drugs, as discussed below. Furthermore, the phenomenon of circadian cycling suggests that inflow is physiologically regulated. However, that regulation seems insensitive to IOP since inflow does not change in glaucomatous patients (Brubaker, 1998). The importance of understanding aqueous humor secretion lies not in clarifying the pathogenesis of glaucoma, but in facilitating development of strategies for lowering IOP. Lowering the IOP is the only intervention as yet documented to delay the onset and reduce the rate of progression of glaucomatous blindness (Collaborative NormalTension Glaucoma Study Group, 1998a,b; The AGIS investigators, 2000; Kass et al., 2002; Leske et al., 2003; Higginbotham et al., 2004). Recent interest has actually focused more on increasing outflow facility (reducing outflow resistance) than on reducing inflow in order to lower IOP, largely because of two theoretical considerations (Gabelt and Kaufman, 2005; Toris and Camras, 2008). First, concern has been expressed about reducing flow

to the avascular anterior segment. However, the baseline flow rate is reasonably rapid, resulting in the total replacement of the ciliary epithelial intracellular fluid in 4 min. This calculation is based on the known area of the rabbit ciliary epithelium (5.72 cm2) [Table I, p. 120 of Cole (1966)] and rabbit inflow [2.72 0.12 ml/min, averaged from data of Table 3 of Toris (2008)], and taking the total height of nonpigmented ciliary epithelial (NPE) and pigmented ciliary epithelial (PE) cells to be 20 mm. Furthermore, as noted above, the physiological circadian reduction in flow during nighttime is actually greater than that achievable with currently available drugs. Second, increasing outflow facility to lower IOP has been thought to be a possibly more physiological strategy since glaucoma is associated with reduced outflow facility and never with increased inflow. However, recent results from studies of the uveoscleral component of total outflow (Gabelt and Kaufman, 2005; Toris and Camras, 2008) raise the possibility that lowering inflow may prove to be the more physiological way to address glaucomatous ocular hypertension. Patients with ocular hypertension display normal inflow rates, but their uveoscleral outflow is reduced by a third (Toris et al., 2002). In order to match outflow to inflow, patients elevate IOP in order to increase outflow through the more pressure sensitive trabecular outflow pathway (Bill, 1966; Toris and Pederson, 1985). The outflow facility of these patients is also reduced by a third (Toris et al., 2002), but it is unclear whether the fall in outflow facility is a cause or a result of the ocular hypertension. It is also unclear whether drugs that increase outflow facility act at the same outflow site aVected in glaucoma. Arguably, it may be more physiological to reduce inflow to match the fall in uveoscleral outflow, rather than stimulate outflow through a pathway possibly diVerent from the physiological routes and diVerent from the site of glaucomatous obstruction.

C. Mode of Aqueous Humor Formation

As recently as 35 years ago, some publications still postulated that the aqueous humor was primarily an ultrafiltrate of the blood (Green and Pederson, 1972). Subsequent data have rendered that view untenable (Krupin and Civan, 1996). From measurements of capillary hydrostatic pressure and stromal oncotic pressure, Bill (1973) concluded that ultrafiltration across the ciliary epithelium would lead to absorption, and not secretion, of aqueous humor. Furthermore, metabolic poisons and selective transport inhibitors such as cardiotonic steroids (Cole, 1960, 1977; Shahidullah et al., 2003) inhibit aqueous humor inflow by 60–80%. In addition, alterations of <25% in systemic arterial pressure about the physiological value have little eVect on

the rate of aqueous humor formation (Bill, 1973; Reitsamer and Kiel, 2008). The higher concentrations of many amino acids (Reddy et al., 1961) and ascorbate in the aqueous humor than in the plasma also indicate that the secretion is transcellular, crossing plasma membranes, and is not simply a largely protein free, paracellular ultrafiltrate.

Although likely of minor direct importance in forming aqueous humor, the arterial pressure is critical for delivering the solutes and water required for transcellular secretion. Progressive reductions by >25% in baseline perfusion pressure or ciliary blood flow lead to progressive falls in aqueous humor secretion (Reitsamer and Kiel, 2003, 2008). The important role of the circulation may also be indicated by the substantially lower net ion (Do and Civan, 2004) and water transfer (Candia et al., 2005, 2007) produced in vitro by iris ciliary bodies isolated from multiple species. In the absence of capillary perfusion, collapse of ciliary processes and a marked increase in unstirred fluid layers would be expected to reduce in vitro secretion. When unstirred layers were minimized by removing the underlying stroma, the isolated rabbit ciliary epithelium was reported to produce a 30 to 50 fold higher rate of net Cl secretion (Crook et al., 2000; Table I). Furthermore, the arterially perfused bovine eye forms aqueous humor at 2.7 0.5 ml/min (Shahidullah et al., 2005), which can be estimated to be approximately threefold higher than that expected from the net Cl flux across the isolated bovine ciliary epithelium (Do and To, 2000).

TABLE I

Cl Fluxes Across the Ciliary Body or Ciliary Epithelial (CE) Bilayer Under Short Circuited Condition

Flux expressed as mEq/h/cm2. Jsa, stromal to aqueous flux; Jas, aqueous to stromal flux. Reprinted (Do and Civan, 2004) with the permission of Springer.

aStatistically significant net Cl secretion.