epithelia. This symport has been immunolocalized at the basolateral surface of PE cells of young calves (Dunn et al., 2001). Inhibition of the symport with furosemide or bumetanide has been found to reduce intracellular Cl activity in shark ciliary epithelium (Wiederholt and Zadunaisky, 1986), reduce Naþ and Cl uptake by cultured bovine PE cells (Helbig et al., 1989a), and shrink native bovine PE cells (Edelman et al., 1994). Blocking the Naþ Kþ 2Cl cotransporter with bumetanide also inhibits net Cl secretion across ciliary epithelium from the rabbit (Crook et al., 2000) and cow (Do and To, 2000), and inhibits aqueous humor formation in isolated, arterially perfused bovine eyes (Shahidullah et al., 2003). In all of these reports, the thermodynamic driving force evidently favored net uptake of Naþ, Kþ, and Cl from the stromal surface into the PE cells. However, the Naþ Kþ 2Cl cotransporter supports bidirectional movement of solute. Reversal of the thermodynamic driving force by reducing ionic concentrations in the bath has been reported to cause bumetanide inhibitable cell shrinkage (Edelman et al., 1994). The strong dependence of the net thermodynamic driving force on intracellular Cl concentration and its implications are considered in greater depth in Chapter 4 (Macknight and Civan, 2008).

b. Parallel Naþ/Hþ and Cl /HCO3 Countertransporters (Antiports)

Measurement of radioactive tracer uptake by cultured bovine PE cells led to the suggestion that Naþ/Hþ and Cl /HCO3 exchange might also be important mechanisms underlying uptake of NaCl from the stroma in vivo (Helbig et al., 1989a; Wiederholt et al., 1991). These antiports were later identified as Naþ/Hþ exchanger NHE 1 and Cl /HCO3 exchanger AE2 by pharmacological and immunostaining approaches, respectively (Counillon et al., 2000). As discussed in Chapter 4 (Macknight and Civan, 2008), electron probe X ray microanalyses have indicated that the antiports are important both on the stromal (Fig. 1A) and aqueous (Fig. 1B) surfaces of intact rabbit ciliary epithelium. Carbonic anhydrase II (CAII) stimulates the turnover of the antiports, both directly and indirectly (Fig. 1A). Intracellular CAII is now known to bind directly to NHE1 (Li et al., 2002) and AE2 (Sterling et al., 2001). CAII also increases the turnover rates of the antiports by catalyzing the production of Hþ and HCO3 from CO2 and water (Meldrun and Roughton, 1933). The importance of CA in catalyzing the turnover rates of the antiports suggests that CA inhibitors act here to reduce inflow and lower IOP.

2. Passage of NaCl from PE to NPE Cells Through Gap Junctions

Gap junctions, considered in depth in Chapter 3 (Mathias et al., 2008), are formed of two hemichannels (half gap junctions or connexons), one at each abutting surface of two adjoining cells. In turn, each connexon consists of six

connexin (Cx) monomers that may be generated from a single connexin (homomeric) or may arise from diVerent connexins (heteromeric). The full gap junction is formed by the linking of connexons of adjoining cells. The full junction may be composed either of identical connexons (homotypic) or of diVerent connexons (heterotypic). Connexin generated gap junctions exclude

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molecules greater than 1 kDa mass, or 6 A radius. The gap junctions may be a site for secretory regulation under certain physiological conditions and could provide a target for pharmacological inhibition.

A great range of techniques has demonstrated the presence of gap junctions linking cells within and between the PE and NPE cell layers, including structural (Reale, 1975; Raviola and Raviola, 1978), biochemical (CocaPrados et al., 1992; Wolosin et al., 1997b; Sears et al., 1998; Do and To, 2000; CoVey et al., 2002; Do, 2002), and functional (Green et al., 1985; Wiederholt and Zadunaisky, 1986; Carre´ et al., 1992; Edelman et al., 1994; Oh et al., 1994; Bowler et al., 1996; Stelling and Jacob, 1997) analyses. Each of the connexin gap junctions thus far identified is both homomeric and homotypic (CoVey et al., 2002). The gap junctions known to link the PE and NPE cells are homomeric, homotypic structures formed from the connexins Cx40 and Cx43, and those known to link adjoining cells in the NPE cell layer arise from connexins Cx26 and Cx31 (CoVey et al., 2002). The molecular basis for the gap junctions linking adjoining PE cells is, as yet, unknown, and might reflect unidentified connexins or the newly recognized, ubiquitous pannexins (Panchin et al., 2000; Panchin, 2005; Barbe et al., 2006; Li et al., 2008). As discussed more fully in Chapter 4 (Macknight and Civan, 2008), the gap junctions linking the PE and NPE cells are more numerous (Raviola and Raviola, 1978) and possibly more robust to certain experimental stresses (McLaughlin et al., 2004) than those linking cells within the PE and NPE cell layers. These observations have led to the view that the PE– NPE cell couplets form the fundamental functional unit of the ciliary epithelium (McLaughlin et al., 2004). The supporting evidence, obtained by electron probe X ray microanalysis, is considered in Chapter 4 (Macknight and Civan, 2008).

The PE–NPE gap junctions are interrupted by the nonselective blockers octanol (Stelling and Jacob, 1997) and heptanol (Mitchell and Civan, 1997). Heptanol also inhibits short circuit current across rabbit (Wolosin et al., 1997a) and bovine (Do and To, 2000) ciliary epithelium and reduces net Cl transport across the bovine preparation (Do and To, 2000). Under baseline conditions, the gap junctions do not likely limit the rate of transcellular NaCl secretion since the elemental compositions of the PE and NPE cells are similar (Bowler et al., 1996). Were the gap junctions to present a substantial barrier under baseline conditions, we would expect to find a higher concentration in the PE cells. However, recent evidence suggests that second messenger

cascades can downregulate solute passage through the PE–NPE gap junctions. Gap junctions are known to be regulated at translational, traYcking, and functional levels (Warn-Cramer and Lau, 2004). However, 30,50 cyclic adenosine monophosphate (cAMP) has been reported to activate Cx40 (van Rijen et al., 2000) but both to increase (Somekawa et al., 2005) and decrease (Lampe and Lau, 2000) communication through Cx43 gap junctions. Transmural measurements of bovine ciliary epithelium have suggested that the overall eVect of cAMP is to block the PE–NPE gap junctions (Do et al., 2004a), a conclusion confirmed by very recent dye transfer and dual cell patch clamping of bovine cell couplets (Do et al., 2008). The multiple roles of cAMP in regulating aqueous humor inflow are further considered in the following sections.

3.Extrusion of NaCl from NPE Cells to Aqueous Humor

a. Naþ, Kþ Activated ATPase. The formation of the aqueous humor ultimately rests upon activity of ciliary epithelial Naþ, Kþ activated ATPase

(Cole, 1960, 1977). Hydrolysis of ATP to ADP is coupled to the extrusion of three intracellular Naþ in exchange for two extracellular Kþ. Thus, ATP utilization provides energy both for secreting Naþ and for establishing the ionic asymmetries and membrane potential needed for secretion of other ions

and of nonelectrolytes.

Although required for secretion, Naþ, Kþ activated ATPase is actually expressed at both surfaces of the ciliary epithelium (Fig. 1A and B). Data obtained by molecular probes (Ghosh et al., 1990, 1991), immunocytochemistry (Mori et al., 1991), and transepithelial electrical measurements (Krupin

et al., 1984) have localized the ATPase to the basolateral membranes of both the PE and NPE cells. In principle, Naþ might be actively transported in opposite directions by the ciliary epithelium toward the stroma and toward

the aqueous humor. Nevertheless, net secretion clearly proceeds from stroma to aqueous humor, and that secretion is strongly inhibited by blocking Naþ, Kþ activated ATPase of the arterially perfused bovine eye with ouabain (Shahidullah et al., 2003). The dominant role of the ATPase of the NPE

over that of the PE cells may reflect at least three factors. First, the number of pumps, assayed by tritiated ouabain binding, is much greater at the aqueous

than at the stromal surface of rabbit ciliary epithelium (Usukura et al., 1988). Second, Naþ, Kþ activated ATPase may be modulated by diVerent regula-

tors in the NPE and PE cells. This possibility is supported by the observation that DARPP 32 (dopamine and cAMP regulated phosphoprotein of Mr

32kDa), a component of phosphorylation mediated modulation of ATPase activity in some cells (Therien and Blostein, 2000), is localized immunohistochemically only to the NPE and not to the PE cells of the rat, cat, rhesus

monkey, and human (Stone et al., 1986). Third, the NPE and PE cell layers express diVerent isoforms of Naþ, Kþ activated ATPase (Martin-Vasallo et al., 1989; Ghosh et al., 1990, 1991; Coca-Prados et al., 1995b; Wetzel and Sweadner, 2001), although the isozyme topography appears to be species dependent (Wetzel and Sweadner, 2001). These isozymes display diVerent ionic binding aYnities and selectivities and diVerent turnover rates (Blanco and Mercer, 1998; Crambert et al., 2000).

The Naþ, Kþ activated ATPase activity of other cells has long been known to be regulated by cAMP activated kinase (protein kinase A, PKA) (Aperia et al., 1991; Therien and Blostein, 2000). For example, ATPase activity of the rat collecting duct was found to be inhibited by a number of agonists that increase cAMP, such as dopamine, vasopressin, and forskolin (Satoh et al., 1993). In part, PKA acts directly by phosphorylating the ATPase at Ser943, thereby reducing its activity. Furthermore, PKA mediated phosphorylation of DARPP 32 inhibits protein phosphatase 1, locking ATPase in a phosphorylated, downregulated state. PKA can aVect ATPase in more complex ways, as well (Therien and Blostein, 2000), by altering the number of plasma membrane pumps, by altering Naþ and Kþ concentrations, by interacting with protein kinase C (PKC), and by activating intermediate proteins. For example, PKA appears to inhibit Naþ, Kþ activated ATPase activity of rat cortical collecting duct by stimulating the cytochrome P450 monooxygenase pathway of arachidonic acid metabolism (Satoh et al., 1993). Whether PKA increases or decreases ATPase activity is species and tissue specific, and depends upon Ca2þ concentration and ROS (Therien and Blostein, 2000). Given these complexities, it is scarcely surprising that reports of the eVects of cAMP on NPE cell Naþ, Kþ activated ATPase have been in incomplete agreement. Administration of db cAMP, a membrane permeant form of cAMP, was found to reduce ouabain sensitive phosphate release from rabbit ciliary epithelium (Delamere and King, 1992). However, the b adrenergic agonist isoproterenol, which increases intracellular cAMP, was reported to increase ouabain sensitive Rbþ uptake by a line of cultured human NPE cells; the b adrenergic antagonist propranolol prevented that stimulation (Liu et al., 2001).

The eVects of PKC, dopamine, and endothelin 1 on NPE cell ATPase have also been complex. For example, activating PKC has stimulated ouabain sensitive Rbþ uptake by a cultured line of rabbit NPE cells (Mito and Delamere, 1993; Delamere et al., 1997). In contrast, PKC activation was reported to inhibit cytohistochemically measured Kþ dependent p nitrophenyl phosphatase in rabbit ciliary epithelium (Nakano et al., 1992).

Divergent results have also been obtained by stimulating NPE cell dopamine (DA) receptors. An agonist of DA1 was found to reduce ouabain sensitive Rbþ uptake by a rabbit NPE cell line (Nakai et al., 1999), but

dopamine did not aVect ouabain sensitive, bumetanide insensitive 86Rbþ uptake by cultured fetal human NPE monolayers (Riese et al., 1998). The diVerent results could have reflected diVerences in cell preparation and experimental conditions. However, the divergence could also reflect the complexity of hormone action. Dopamine is thought to aVect Naþ, Kþ activated ATPase activity of other cells through both DA1 and DA2 receptor stimulated, PKC dependent mechanisms and DA1 stimulated, PKA associated pathways (Therien and Blostein, 2000).

Endothelin 1 also exerts complex eVects on the NPE cells. The hormone produced a direct inhibition of enzyme activity, but also increased mRNA for its synthesis in transformed human NPE cells (Krishnamoorthy et al., 2003).

The second messenger nitric oxide (NO) also reduces ouabain sensitive Naþ, Kþ activated ATPase activity of native porcine NPE cells (Shahidullah and Delamere, 2006). The inhibition is observed whether NO is delivered by donor molecules or generated by nitric oxide synthase (NOS). In contrast, NOS generated NO has recently been reported to stimulate Naþ, Kþ activated ATPase activity of rabbit cardiac myocytes, measured as whole cell, electrogenic Naþ Kþ pump current (White et al., 2008).

In summary, multiple hormones and second messenger cascades modulate Naþ,Kþ activated ATPase activity of the ciliary epithelium, but their actions can be direct or indirect, and depend on isoform specificity and interactions with parallel signaling cascades. A further complexity arises from increasing evidence that Naþ,Kþ activated ATPase itself plays a key role in signaling cascades, which is independent of its eVects on intracellular Naþ and Kþ concentration (Xie and Askari, 2002). This newly appreciated role includes eVects on gene regulation and cell growth, mediated through protein–protein interactions.

b. Cl Channels. Extrusion of Naþ through Naþ, Kþ activated ATPase is accompanied by release of Cl into the aqueous humor through anion channels of the NPE cells. Several observations suggest that this release is a rate limiting factor in aqueous humor formation. Of the three steps comprising aqueous humor formation, stromal uptake of NaCl is not rate limiting under baseline conditions since the PE cell Cl concentration is fourfold higher than that expected at electrochemical equilibrium. As noted in Section IV.B.1, this relatively high intracellular Cl concentration is established by the electroneutral symports and antiports of the PE cells. The second step, transfer of NaCl, from the PE to NPE cells, is also not likely rate limiting since the Cl contents (McLaughlin et al., 2007), Cl concentrations (Bowler et al., 1996), and intracellular potentials (Green et al., 1985) of the two cell layers are closely similar. By exclusion, the aqueous surface of the ciliary epithelium is likely to be the major site of regulation. As discussed

in Section IV.B.3.a, Naþ, Kþ activated ATPase at this surface can certainly be modified, but its continuous activity, necessary for maintenance of transmembrane ionic asymmetries, is readily detected under baseline conditions (Krupin et al., 1984). In contrast, Cl channel activity of native bovine NPE cells is low under baseline conditions, and can be enhanced by a number of perturbations (Section VI).

The molecular identity of Cl channels at the aqueous surface has not yet been established. More than one channel is likely expressed since hypotonic swelling of native bovine NPE cells was found to activate Cl channels with unitary conductances of 7.3 and 18.8 pS (Zhang and Jacob, 1997). Several lines of evidence have suggested that ClC 3 (Coca-Prados et al., 1996; Civan, 2003) or pICln (Anguı´ta et al., 1995; Coca-Prados et al., 1995a) might play substantial roles in NPE cell Cl channel activity.

ClC 3 has been implicated by the observations that: (1) NPE cells express ClC 3 transcripts and protein product (Coca-Prados et al., 1996; Sanchez Torres et al., unpublished observation); (2) activation of PKC lowers NPE cell Cl channel activity (Civan et al., 1994; Coca-Prados et al., 1995a, 1996; Shi et al., 2003; Do et al., 2005), a signature property of Cl currents associated with ClC 3 (Kawasaki et al., 1994); (3) antisense oligonucleotides knockdown ClC 3 message and protein product in NPE cells, and also reduce volume activated Cl currents (Wang et al., 2000); and (4) blocking antibody directed against ClC 3 (Wang et al., 2003) reduces swelling activated Cl currents of both transformed rabbit NPE cells (Vessey et al., 2004) and native bovine NPE cells (Do et al., 2005). These results link ClC 3 to Cl channels, but its precise role is unclear, both in the NPE and other cells. Whether ClC 3 is necessary for expression of swelling activated Cl channels in any cell has been controversial (Hermoso et al., 2002; Jentsch et al., 2002). At issue has been whether swelling activated Cl channels in other cells of ClC 3 null mice are diVerent from those of the wild type mice (Stobrawa et al., 2001; Gong et al., 2004; YamamotoMizuma et al., 2004; Wang et al., 2005). Another issue has been whether ClC 3 is a Cl channel, like ClC 1, ClC 2, ClC Ka, and ClC Kb, or whether ClC 3 functions as a Cl /Hþ antiport exchanger, like ClC 4, ClC 5, and the bacterial homologue ClC ec1 (Jentsch, 2007; Zifarelli and Pusch, 2007). One possible interpretation is that ClC 3 may form part of a protein complex constituting the swelling activated Cl channel. Another possibility is that ClC 3 plays roles in the posttranslational processing, traYcking, and/or regulation of other swelling activated Cl channels. The latter possibility is consistent with the observation that PKC activation initially inhibited swelling activation of NPE cell Cl channels, but did not aVect steady state activation (Do et al., 2005). Among other interpretations that result may reflect a role of ClC 3 in the traYcking or regulation of diVerent Cl channels capable of mediating swelling activated Cl channels.

Substantial experimental work has also raised the possibility that pICln (Paulmichl et al., 1992) might underlie or regulate swelling activated NPE cell Cl channels. pICln is not only found in, but its human form was first cloned from, the NPE cells (Anguı´ta et al., 1995; Coca-Prados et al., 1995a). Furthermore, an antisense oligonucleotide directed against pICln downregulated both protein and swelling activated Cl currents in native bovine NPE cells (Chen et al., 1999). Nevertheless, as for ClC 3, the potential role of pICln in expressing swelling activated NPE cell Cl currents has been, and remains, controversial (Clapham, 1998; Strange, 1998; Fu¨rst et al., 2006). At issue have been the questions whether pICln is physiologically present in the plasma membrane, whether it functions as a channel, and if so, whether its selectivity conforms to a Cl channel. The question has even been raised that the role of this ubiquitous, abundant, and conserved protein may not be directly related to swelling activation of Cl currents in other cells (Strange, 1998). In the case of the NPE cells (Sanchez-Torres et al., 1999), pICln was immunolocalized to the cytoplasm and perinuclear region and was not translocated to the plasma membrane by hypotonic challenge. These results have suggested that the functional eVects of antisense knockdown of pICln (Chen et al., 1999) may be mediated indirectly, possibly through restructuring of the cytoskeleton.

c. Kþ Channels. Kþ channels subserve at least three main functions. In addition to providing a pathway for release of Kþ down its electrochemical gradient to the aqueous humor (Fig. 1A), these channels are needed to maintain the intracellular potential more negative than the Cl equilibrium (Nernst) potential. The more negative the intracellular potential, the greater is the thermodynamic force driving Cl secretion. The third function of the Kþ channels is to provide a conduit for Kþ to act as a catalyst, enhancing physiological turnover of other transporters. At the basolateral surface of the NPE cells (Fig. 1A), release of intracellular Kþ ensures a high enough extracellular Kþ concentration to support rapid cycling of the Naþ, Kþ exchange pump. At the stromal surface, Kþ channels (Fig. 1B) ensure that the Kþ concentration is high enough to help drive NaCl into the PE cell through the Naþ Kþ 2Cl symport. In either case, the Kþ channels act to accelerate cycling either of the symport and/or of Naþ, Kþ activated ATPase. This function is particularly well illustrated by the loss of function mutation of the luminal ROMK2 Kþ channel that interferes with symport uptake of Naþ Kþ 2Cl by the thick ascending limb of the renal loop of Henle, producing one form of Bartter’s syndrome with urinary loss of salt and volume depletion (Hebert, 2003).

Both the NPE and PE cells express multiple Kþ channels, including inward rectifiers, delayed outward rectifiers, and Ca2þ activated outward rectifiers (Jacob and Civan, 1996; Bhattacharyya et al., 2002). Inward rectifiers pass