The Role of Gap Junction Channels in the Ciliary Body Secretory Epithelium

apical surfaces appose one another. At the apical–apical interface, the two layers are in communication through gap junctions formed from connexins Cx40 and Cx43. Cells of the PE are not in direct gap junctional communication with each other; however, the cells of the NPE are coupled by gap junctions made from Cx26 and Cx32. We first review the properties of the ciliary body, then those of gap junction channels in general, with special emphasis on channels made from the connexins present in the ciliary epithelium. An important question arises concerning the gap junction channels at the apical–apical interface: Do these channels conduct the water that is ultimately secreted? Model calculations are presented that suggest the channels could do so, but only if they provide a rather high degree of ion coupling. Finally, experimental animal models, which might help test this hypothesis, are reviewed.

II. INTRODUCTION

A. The Anatomy of the Ciliary Body

The ciliary body is an annular structure that attaches to the lens via the suspensory ligament and is connected to the sclera at the base of the cornea. The major tissue type within the ciliary body is smooth muscle, which has a role in the accommodation process. On the surface of the ciliary body, facing the posterior chamber, a number of fingerlike projections are found. These make up the secretory epithelium of the ciliary body. Each process has an underlying, capillary rich, connective tissue that is covered by a stratified cuboidal epithelium. The stratified epithelium represents the site of the blood–AH barrier. The epithelium consists of two cell layers: a pigmented layer that faces the interstitial and vascular space, and a nonpigmented layer that faces the posterior chamber. The apical surfaces of the pigmented cells are in intimate contact with the apical surfaces of the nonpigmented cells.

B. Ciliary Body Epithelial Function: Production of Aqueous Humor

The ciliary body secretory epithelium generates a slightly hyperosmotic to isosmotic fluid of 300 mOsm (Hayward et al., 1976; Gaasterland et al., 1979) and is able to generate fluid flow rates from plasma to aqueous of 15–30 ml/hour in animal models (Candia et al., 2005). Detailed experimental analysis and modeling of the stratified epithelium suggest the net flux of chloride and sodium ions as the motive force in generating the AH. The

transport of fluid from the ciliary body into the posterior chamber is balanced by the drainage of fluid by Schlemm’s canal. The autonomic nervous system is the major modulator of fluid transport and drainage (Uusitalo, 1972).

Various ion channels and transporters (Do and Civan, 2006) participate in the extraction of ions from the vascular space and subsequent transport to the AH. Water is thought to follow as a consequence of osmosis and is facilitated by the presence of aquaporins (Patil et al., 1997). Gap junction channels are unique within the ensemble of channels and transporters (CoVey et al., 2002). They are unique because they neither extract nor secrete from either epithelial surface. Rather they are presumed to allow the stratified epithelium to act as a monolayer, facilitating the movement of solutes from the pigmented to the nonpigmented layer. The blood–AH barrier is associated with the presence of tight junctions on the lateral surfaces of NPE cells. Given this anatomical barrier, the transepithelial movement of ions and other solutes is thought to be via the gap junction channels connecting the two cell layers. Pharmacological inhibition of gap junction channel activity reduces the net flux of ions across the epithelium (Wolosin et al., 1997; Do and Civan, 2004), supporting the role of gap junctions in transepithelial solute flux.

These data implicate gap junctions as a necessary component of the ciliary body; gap junctions create a functional syncytium connecting the PE and NPE. The end result of such a linkage is a double layered epithelia functioning similar to a simple monolayer with regard to ions and small molecules. To gain a better understanding of how gap junction channels within the ciliary body epithelium might participate in the production of AH, we will review first the subunit proteins forming the gap junction channels, second the distribution of diVerent channel types, and third the functional properties of those gap junction channels both in general and in relation to the ciliary body. We shall seek to answer the question: are the known properties of ciliary body gap junctions of importance for the normal functioning of the epithelium?

C. Gap Junction Channels Formed by Connexins

Gap junction channels in vertebrates are formed from subunit proteins called connexins. A gap junction channel is composed of two hemichannels, each of which is formed from six connexins. When two cells are in close apposition, it is possible for a hemichannel from each cell to link together via the extracellular loops of the component connexins to form a cell to cell gap junction channel. This channel represents a unique intercellular pathway

because it is the only form of intercellular communication that excludes the extracellular space. Gap junction channels tend to aggregate and form plaques containing tens to thousands of channels (Goodenough, 1975). The reasons for plaque formation are not completely understood, but have been attributed to lipid membrane domains including lipid rafts (Locke et al., 2005).

There are over 20 identified connexins within the human genome and all are able to form gap junction channels between cells. Most cell types coexpress several connexins. For example, in the heart almost every cell type expresses at least two of the following three connexins, Cx40, Cx43, and Cx45 (Van Veen et al., 2001), while liver hepatocytes and the lacrimal gland coexpress Cx26 and Cx32 (Kojima et al., 1996; Walcott et al., 2002; Ott et al., 2006). The possibility therefore exists of channels being formed that contain more than one connexin type. If a hemichannel contains two connexin types and forms a gap junction channel with an adjacent cell also containing hemichannels made from two connexin types, then the channel is called a heteromeric. If all the subunit connexins from both cells are only of one type of connexin, then the channel is referred to as homotypic. If two adjacent cells are each making a single type of hemichannel, but by diVerent connexins, then cell to cell gap junction channels are referred to as heterotypic. A number of connexins have been shown to form heteromeric and heterotypic gap junction channels (Brink et al., 1997; Valiunas et al., 2000, 2001; Cottrell et al., 2002), but not all connexins are able to mix (White and Bruzzone, 1996; Gemel et al., 2004).

D. Connexins in the Ciliary Body Epithelium

The ciliary body is no exception to the general rule that tissues express more than one connexin. In fact, four connexins are expressed in the nonpigmented cell layer whereas two are expressed in the pigmented layer (CoVey et al., 2002). The PE and NPE both express Cx40 and Cx43, which colocalize on their apposing apical surfaces (CoVey et al., 2002). This colocalization implies that these connexins could be forming mixed channels, but an explicit demonstration of heteromeric or heterotypic channels between these two cell types has not been shown. A number of studies using Cx40 and Cx43 have shown that these two connexins have the ability to form heteromeric and heterotypic channels (He et al., 1999; Valiunas et al., 2001, 2002), but other studies suggest that they prefer to not to do so (Bruzzone et al., 1993; Rackauskas et al., 2007). In addition, the cells of the NPE couple to each other with gap junction channels composed of either Cx26 or Cx31 (Fig. 1).