III. STRUCTURE OF CILIARY EPITHELIUM

The ciliary epithelium that covers the ciliary body consists of a major pars plicata anteriorly and a minor pars plana posteriorly in the human. The pars plicata is composed of 70 villiform processes extending anteriorly to the pupil. Connective tissue, vessels, and nerve endings comprise the stroma of each process. The pars plana is flat and extends posteriorly to the ora serrata, the demarcation with the neuroretina (Pei and Smelser, 1968).

As a result of the embryological invagination of the optic vesicle to form the optic cup, the microanatomy of the ciliary epithelium is unique (Krupin and Civan, 1996). Unlike other epithelia, the two cell layers adjoin each other at their apical surfaces (Fig. 1). The basolateral surfaces of the outer PE cells abut the stroma and those of the inner NPE cells face the aqueous humor. Gap junctions provide low resistance pathways interconnecting the intracellular fluids of cells within and between the two cell layers (Raviola and Raviola, 1978). The gap junctions of the ciliary epithelium are considered in depth by Mathias et al. (2008; Chapter 3) in this volume.

IV. UNIDIRECTIONAL SECRETION OF AQUEOUS HUMOR

A. Basic Strategy of the Ciliary Epithelium

1. Relationship of Solute and Water Secretion

Secretion has long been thought to be based upon a primary transfer of net solute from stroma to aqueous humor, thereby establishing an osmotic gradient. Water has been considered to follow secondarily by local osmosis. The discovery of aquaporin (AQP) water channels has unequivocally demonstrated that water movement can indeed be dissociated from solute movement in response to local osmotic gradients (King et al., 2004). AQPs are considered briefly in a later section and in depth in Chapter 2 (Stamer et al., 2008).

Whether all transmembrane water movement proceeds through local osmosis has recently been questioned (Loo et al., 2002; Fischbarg et al., 2006). A series of publications has reported evidence suggesting that water may also be transferred across biological membranes in fixed stoichiometry to ions and nonelectrolytes simultaneously cotransported (Loo et al., 2002). Whether water is ever cotransported at a fixed stoichiometry, and if so, whether it is quantitatively significant, has been controversial (Lapointe, 2007; Zeuthen and Zeuthen, 2007). In addition, electroosmosis has long been considered a possible contributor to transepithelial water movement (McLaughlin and

H2O

FIGURE 1 Transport components underlying unidirectional secretion (A) and possible unidirectional reabsorption (B) across the ciliary epithelium. Tight junctions (tj) between the NPE cells provide a barrier between the stromal and aqueous compartments. Gap junctions (gj) subserve intercellular communication between adjoining PE cells, NPE cells, and PE–NPE cell couplets. Carbonic anhydrase (CA) directly stimulates the Naþ/Hþ and Cl /HCO3 antiports.

Mathias, 1985), a possibility readdressed in a recent series of studies (Fischbar get al,. 2006). The ana lysesLapoiof nte (2007)and Ma thias and Wang (2005) raise doubt whether it is necessary to invoke either water cotransport or electroosmosis, respectively, to account for transepithelial water movement. The current prevailing view is that transepithelial water flow generally proceeds by local osmosis. ATP is expended in order to transfer solute across epithelia in order to establish an osmotic gradient for secondary, uncoupled secretion of water.

2. Centrality of NaCl Secretion

As noted above (Sections II.A and C), the composition of the aqueous humor diVers from that of the plasma. Nevertheless, both plasma and aqueous humor are largely solutions of NaCl, with Naþ and Cl concentrations of 150 and 130 mM, respectively, in the human aqueous humor (Krupin and Civan, 1996). Thus, the formation of the aqueous humor can be viewed essentially as a primary, energy dependent transfer of NaCl, and a secondary transfer of water, across the ciliary epithelium. Consistent with this view, blocking Naþ or Cl transepithelial transport reduces the rate of aqueous humor formation (Shahidullah et al., 2003). The minor constituents of the stromal extracellular fluid and aqueous humor, especially HCO3 , Kþ, and Ca2þ, are known to modulate secretion, but those important eVects are exerted indirectly on Naþ and Cl transfer (Krupin and Civan, 1996; To et al., 2001; Do and Civan, 2004).

3. Transcellular and Paracellular Components of Secretion

In principle, solutes and water can be transferred through both the transcellular pathway through the cells and the paracellular pathway between the epithelial cells. Taking the convoluted surface of the isolated, full thickness ciliary epithelium into account, the transmural resistance of the rabbit preparation is 1 KO cm2 (Krupin et al., 1984). However, much of that resistance may reflect contributions of the stroma underlying the epithelium. After isolating small areas of the rabbit epithelial bilayer, Sears et al. (1991) found that the transmural resistance was reduced to 40 O cm2, even though the transepithelial potential was still 0.65 mV, a value comparable to that measured across the full thickness preparation (Krupin and Civan, 1996). This purely transepithelial resistance corresponds to that of a leaky epithelium (Rose and Schultz, 1971; Fro¨mter and Diamond, 1972), suggesting that the paracellular pathway provides a substantial transmural electrical shunt. The observation that transport inhibitors can reduce inflow in experimental preparations by as much as 60–80% indicates that the secretory pathway is largely transcellular. However, the isolated ciliary epithelium of multiple species displays a transepithelial potential diVerence of 1 mV, with the

aqueous humor negative to the stroma. Whether this small driving force produces a significant paracellular Naþ contribution to total secretion is unknown.

Transcellular epithelial transfer of NaCl fundamentally depends on direct coupling of ATP utilization with Naþ movement through Naþ, Kþ activated ATPase, but is also mediated by an ensemble of ion and water channels, cotransporters (symports) coupling flows in the same direction, and countertransporters (antiports) coupling solute flows in opposite directions. These transport components are introduced in the following section. A comprehensive discussion of the ion channels described in the following section is provided in the monograph by Hille (2001).

B. Transport Components Underlying Transcellular Secretion

The ciliary epithelium expresses a wide range of ion channels and transporters responsible for facilitated diVusion, cotransport, and countertransport (Jacob and Civan, 1996; Krupin and Civan, 1996). Many of these transport elements perform housekeeping tasks necessary for individual cell viability and function. This chapter focuses on the channels and transporters likely to be directly involved in transepithelial secretion of ions and water (Fig. 1A).

1. Uptake of Stromal NaCl

The first step in transepithelial secretion is the uptake of NaCl from the stromal extracellular fluid by the PE cells (Fig. 1A). The intracellular potentials of the PE and NPE cells are very similar to each other and highly negative (Green et al., 1985), so that the electroneutral transporters indicated in Fig. 1A permit the PE cells to take up Cl against a strong electrochemical gradient. Measured with the same extracellular bathing solution containing 152 mM Cl , the intracellular potential of rabbit ciliary epithelium was found to be 67.0 0.2 mV (N ¼ 110) (Carre´ et al., 1992), and the intracellular Cl concentration was estimated by electron probe X ray microanalysis to be 46 5 mmol/kg intracellular water (N ¼ 99) (Bowler et al., 1996). This value is fourfold higher than the intracellular Cl concentration calculated for an equilibrium distribution at the measured membrane potential. At least two sets of electroneutral transporters support uptake of NaCl from the stroma (Wiederholt et al., 1991) as described below.

a. Naþ Kþ 2Cl– Cotransporters (Symports). Following the report by Geck et al. (1980), the Naþ Kþ 2Cl cotransporter has been identified as a major mechanism for uptake of NaCl by both secretory and absorptive