Despite the possibility of other mechanisms, the analysis presented here suggests that gap junctions can indeed transport significant water flow and thus are the prime candidate for the path leading to AH formation.

V.ANIMAL MODELS SUPPORT A ROLE FOR GAP JUNCTIONS IN FLUID TRANSPORT BY OCULAR EPITHELIA

Having reviewed the functional properties of ciliary body gap junctions and then hypothesizing how those properties could participate in secretion of the AH, we will now evaluate evidence from genetically engineered mice to see if the available experimental data are consistent with our model.

A recent study by Calera et al., (2006) examined mice in which the Cx43 protein was eliminated by conditional gene knockout in the ciliary body. Immunohistochemical staining showed that Cx43 was eliminated from the PE but not the NPE, resulting in a ciliary epithelium that displayed areas of separation between the pigmented and nonpigmented layers. By 2 weeks of age, knockout mice had smaller eyes that were flaccid when dissected. The authors speculated that these flaccid eyes were due to a reduction in IOP, which in turn was due to a reduction in ion and water flow across the ciliary epithelium caused by the loss of Cx43 in the PE. Although IOP was not measured in this study, the authors provided some support for this hypothesis by showing that back diVusion of plasma proteins into the AH was occurring in the conditional knockouts, leading to protein precipitates in the anterior chamber. By 5 weeks of age, the mice had a dramatically reduced vitreal space and a variety of other ocular defects. Due to the choice of Cre expressing mice (nestin Cre) used to generate these animals, Cx43 was removed from a number of ocular cell types in addition to the PE, complicating interpretation of the described phenotype. While further study will be required, it is clear that loss of Cx43 in the PE was correlated with a loss of morphologically recognizable gap junctions from the NPE/PE when examined by electron microscopy. The observed elevation in plasma protein in the AH and pathohistological changes consistent with loss in IOP are consistent with the model for fluid secretion described above.

A second example of an epithelium where gap junction channels and their distribution have been shown to aVect the secretion of fluid is provided by the lacrimal gland, where a monolayer of acinar cells generates the fluid of tears. These cells express Cx32 and Cx26. In Cx32, knockout animals tear production was significantly reduced in female, but not male mice (Walcott et al., 2002). The distribution of Cx26 in the Cx32 knockout mice was also determined and in the males extensive Cx26 containing gap junction plaques were found but in the females Cx26 staining was absent from the plasma

membrane (Walcott et al., 2002). Thus lateral coordination of lacrimal epithelial function was lost in the females and this disrupted fluid secretion. Gap junctions can therefore aVect fluid transport either directly, by actually being the path of fluid flow as in the model in this review, or indirectly through signaling between cells.

The lens of the eye generates an internal circulation of ions and fluid (reviewed in Mathias et al., 2007). This circulation enters the lens through the extracellular spaces between cells, moves into fiber cells, and then circulates to the equatorial surface cells by flowing through fiber cell gap junctions made from Cx46 and Cx50. Knockout of Cx46 (Gong et al., 1997) caused loss of coupling between the central mature fiber cells (Gong et al., 1998). This disrupted the egress pathway for the lens’ circulation and caused calcium to accumulate in the central fiber cells (Gao et al., 2004). Disruption of the path for ion flow would be suYcient to disrupt the circulation of fluid, so these data do not directly demonstrate a role of gap junctions in conducting fluid flow in the lens, but they are consistent with such a role.

There are additional connexin knockout/knockin mice that would be potentially useful for evaluating the role of gap junctions in the ciliary body. As noted above, Cx40 knockout animals might be expected to have diminished production of a hypertonic AH, which could be experimentally tested as these mice are viable (Simon et al., 1998). Perhaps more intriguing would be knockin mice where the Cx43 gene has been replaced with either Cx32, Cx40, or Cx26 (Plum et al., 2000; Winterhager et al., 2007). These mice would explore the role of connexin specificity in AH production and allow comparison of the roles of relatively nonspecific ionic coupling (provided by all connexins) with the selective permeability for larger solutes such as cyclic nucleotides (which diVer dramatically from one connexin to another), and perhaps water permeability, which potentially could vary between connexins. Many of the ideas expressed in this chapter could be easily tested with existing animal models.

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I. OVERVIEW

The ciliary epithelium secretes the aqueous humor into the posterior chamber of the eye. Despite the physiologic and pharmacologic importance of this epithelium, its complex structure and heterogeneous cell composition have impeded progress in studying the integrated functioning of the intact tissue. Electron probe X-ray microanalysis has provided an unusual opportunity to study the different cell populations in different regions of the intact ciliary epithelium. This chapter summarizes the advances in our understanding made possible by exploiting this technique.

II. INTRODUCTION

The mammalian ciliary epithelium is often studied as though it were a homogeneous preparation. However, both anatomical and histological studies show significant diVerences between diVerent regions. The minor, flat pars plana is posterior to two well defined anatomic regions, the posterior and anterior portions of the ciliary epithelium. The posterior region, the posterior pars plicata, displays long ciliary processes reaching regularly down to the iris. In contrast, in the rabbit anterior region, comprising the iridial portion of the primary ciliary processes (Weingeist, 1970), the folds are more tortuous. From histological studies, it is known that proteins and biologically active peptides are expressed nonuniformly in diVerent regions of the ciliary epithelium (Flu¨gel and Lu¨tjen Drecoll, 1988; Flu¨gel et al., 1989, 1993; Eichhorn et al., 1990; Ghosh et al., 1990, 1991; Eichhorn and Lu¨tjen Drecoll, 1993; Dunn et al., 2001). However, a number of investigators have observed regional diVerences in the expression of Naþ, Kþ activated ATPase (Ghosh et al., 1990) and additional proteins and biologically active peptides. For example, nonpigmented epithelial (NPE) cells of young calves display higher expression of a1/a2/a3/b1/b2 isoforms of Naþ,Kþ activated ATPase anteriorly than posteriorly, but pigmented epithelial (PE) cells expressed a constant relative concentration of a1/b1 throughout the epithelium (Coca Prados and Sa´nchez Torres, 1998). Another example is the localization of the Naþ Kþ 2Cl cotransporter largely at the basolateral edge of the PE cell layer in the anterior region of young calves (Dunn et al., 2001).

Until recently, however, it has not been possible to assess the functional significance of these variations in ciliary epithelial organization. Much has been learnt about overall aqueous humor secretion with isolated ciliary bodies from a variety of mammalian species studied in Ussing chambers under open and short circuited conditions. However, electrical

measurements do not detect electroneutral solute movements and, given the leaky nature of the tissues, it is not always easy to quantify cellular solute movements from isotope studies. Other techniques that have been employed include microelectrodes, patch clamping, and fluorescence microscopy with isolated cells or groups of cells. None of these, however, enables one to determine the cellular ion composition of the transporting cells in an intact ciliary body. Without this, it is not possible to identify the ions and describe the major pathways involved in aqueous humor production.

We have applied the technique of electron probe X ray microanalysis (EPMA) to study this problem. This approach allows measurement of the elemental composition of individual epithelial cells within the intact tissue and has provided new insights into the transport properties of the ciliary epithelium.

III.REVIEW OF ELECTRON PROBE X RAY MICROANALYSIS

A. Theory

EPMA permits localization and quantification of intracellular elements within visualized cells. Using an electron microscope, a specific area within a cell is targeted with an electron beam. Incident electrons of suYcient energy can knock electrons out of the inner shell of an atom within the irradiated area. When an electron from an outer orbit relaxes into the vacated orbit, a quantum of X ray energy is released that is characteristic of the atom bombarded. Measurement of the number of quanta at each characteristic energy permits quantification of the identified intracellular elements.

B. Technique

The application of this technique to cells requires the tissue to be mounted in an electron microscope with the elements remaining in the positions that they occupied in the living state. It has been shown that the cellular and extracellular locations of diVusible ions can be preserved by a combination of very rapid freezing of tissues to liquid N2 temperatures followed by thin sectioning of the frozen tissues in a cryoultramicrotome at temperatures below –80 C. The sections 0.2–0.4 mm in thickness must then be freeze dried at these temperatures and at subatmospheric pressures (typically around 10 4 Pa or 7.5 10 7 Torr) to remove the water while preserving cell elemental contents.

The frozen dried sections can then be visualized in a scanning electron microscope with a transmitted electron detector. X rays are collected, usually with an energy dispersive X ray detector. The intracellular data are obtained by scanning the electron beam over a rectangular area within each selected cell. It is important to avoid the cell boundaries as, during the freeze drying, extracellular elements tend to collect at the cell surface so that a cellular signal acquired near the plasma membrane often has artificially high Naþ and Cl contributions. For this reason, we normally choose to sample an area within the nucleus. Direct measurements of Chironomus salivary gland cells have demonstrated that the intracellular activities of Kþ and Cl are the same in the nucleoplasm and cytosol (Palmer and Civan, 1975, 1977). In practice, the dimensions of the irradiated areas vary from 0.9 1.2 mm to 2.4 3.0 mm, depending on the size of the nucleus analyzed.

Elemental peaks can be quantified by filtered least square fitting to a library of monoelemental peaks (Bowler et al., 1991). The library spectra for Na, Mg, Si, P, S, Cl, K, and Ca are derived from microcrystals sprayed onto a Formvar film. In addition to the quantal element specific X rays, irradiating sections with an electron beam produces nonquantal continuous or white radiation, reflecting electron deceleration by coulombic interaction with atomic nuclei. The white counts (w), an index of tissue mass (Civan, 1983), are summed over the range 4.6–6.0 keV, and corrected for the nontissue contributions arising from the specimen holder and grid.

C. Application to the Ciliary Epithelium

In our studies, we use tissues from adult Dutch black belted rabbits of either sex. The iris ciliary body (ICB) is excised from each enucleated eye, cut into quarters, and each quarter bonded with cyanoacrylate to a Mylar support frame on its stromal border.

Quadrants of each ICB are mounted between the two halves of incubation chambers, so oriented as to occlude the common aperture. Each half chamber is filled with 1.5 ml, and fresh solution constantly infused at 0.5 ml/min. A gas lift in each half chamber aerates and gently stirs the solution. Drugs, when used, are normally added to both sides of the tissue to maximize the eVects.

Tissues are incubated for 1–2 hours at room temperature (18–22 C), initially under control conditions. Pairs of quadrants (one from each eye) are then incubated for at least 30 min under either control or experimental conditions. After incubation, the tissues are blotted, and then plunged into liquid propane at –180 C to freeze the preparation rapidly and preclude redistribution of ions and water. Blocks are fractured from the frozen tissue

CO2 + H2O

Stroma

PE cells

FIGURE 3 Stimulation of KCl uptake by CO2/HCO3 . HCO3 and Hþ are formed from water and CO2 through a reaction catalyzed by the enzyme carbonic anhydrase (CA). Both Cl /HCO3 and Naþ/Hþ exchangers operate on the basolateral membrane of the PE cells and remove HCO3 and Hþ from the cells, which gain Naþ and Cl in exchange. This Naþ is then extruded from the NPE and PE cells by the Naþ,Kþ activated ATPase, both at the stromal surface (illustrated here) and at the aqueous surface. The net eVect is a gain of Cl with Kþ by the cells.

et al., 1985). Likewise, we can use the normalized anion gap, defined as (NaþK Cl)/P, as an approximate index of changes in intracellular HCO3 content (McLaughlin et al., 2001b), although other unmeasured anions can, of course, also contribute to this parameter.

IV. TOTAL INFLOW

A. Feasibility of EPMA

It was first necessary to establish that the technique of EPMA could be applied to the ciliary epithelium. We initially demonstrated that loss of Kþ and gain of Naþ after tissue exposure to ouabain was readily detected and then went on to study cell elemental composition under a variety of situations.

B. Role of Gap Junctions Between PE and NPE Cells

We found that, under control conditions, there were no measurable elemental gradients across the gap junctions between paired PE and NPE cells for Naþ, Kþ, or Cl , when allowance is made for the diVerences in P content between the two cell types. This indicates that, under normal conditions, the gap junctions between paired PE and NPE cells do not represent a rate limiting step in transepithelial ion movements. When we used heptanol to block the gap junctions, at least partially, the elemental contents of the two cell types did not change appreciably unless transport inhibitors, such as ouabain, were used (McLaughlin et al., 2004). This finding is consistent with our understanding that, when the cells’ multiple transporters are all available, cells adjust the rates of movement of ions to maintain their steady state compositions.

C. Cellular Chloride

Chloride plays a key role in transepithelial secretion and absorption of salt and water. For example, loss of function mutation of the Clcnk gene for the ClC Kb Cl channel produces the type III form of Bartter’s syndrome, with renal salt wasting (Hebert, 2003). However, an integrated model of the many mechanisms underlying net ciliary epithelial Cl secretion and its regulation has not been fully developed.

The electron microprobe was first utilized to study the known stimulatory eVects of CO2/HCO3 on the rabbit ciliary epithelium. Exposure to CO2/HCO3 increases the magnitude of the intracellular ciliary epithelial potential (Carre´ et al., 1992), restores the normally negative transepithelial potential (aqueous with respect to stroma) (Kishida et al., 1981; Krupin et al., 1984), and stimulates transepithelial secretion of fluid (Candia et al., 2005). We demonstrated that the presence of CO2/HCO3 in the bath resulted in increased cell Cl content, together with an increase in cell Kþ content, which was prevented by the carbonic anhydrase inhibitor acetazolamide (Fig. 2). The increase in Cl content likely reflects exchange for intracellular HCO3 through a Cl /HCO3 antiport. The increase in Kþ content can be understood as arising from two sequential steps (Fig. 3), an initial exchange of extracellular Naþ for intracellular proton through a Naþ/Hþ antiport, and subsequent exchange of intracellular Naþ for extracellular Kþ through Naþ, Kþ activated ATPase.

Another widely distributed Cl transport pathway, the Naþ Kþ 2Cl cotransporter was also studied. In secretory epithelia, it is this transporter that is usually responsible for accumulation of Cl to levels greater than would

D.Role of the Naþ, Kþ Activated ATPase in Aqueous Humor Production in Rabbit Ciliary Epithelium

Naþ,Kþ activated ATPase is found on the basolateral membranes of both the PE and NPE cells. We have studied the eVects of inhibiting the pump with the cardiac gycoside ouabain, both in the absence and presence of the gap junction uncoupler heptanol. We have found that ouabain causes significant loss of Kþ and uptake of Naþ by the cells (Fig. 7). With ouabain only on the

FIGURE 7 The eVects of 0.1 mM ouabain on the ion contents of cells incubated in CO2/ HCO3 Ringer’s solution (McLaughlin et al., 2004). In these box plots, the medians are indicated by the central horizontal lines, the notch indicates the 95% confidence intervals, the lower and upper lines include all data between the 25th and 75th percentiles, and the ‘‘whiskers’’ display the data range between the 10th and 90th percentiles. Changes in Naþ and Kþ are far greater in the anterior than posterior region. Since the rate limiting step in cation changes after ouabain is the membrane Naþ permeability, the anterior cells must have a higher Naþ permeability than the posterior cells. Used with the permission of the American Physiological Society.