Water Channel Regulatory Gene(s) in Ciliary Epithelia

One of the main treatments of glaucoma is the use of drugs that decrease the secretion of aqueous humor fluid from the region of the eye known as the ciliary epithelium, in order to lower intraocular pressure. Unfortunately, many currently available drugs that decrease aqueous humor production such as adrenergic antagonists, may cause serious systemic side effects such as cardiac arrhythmias and arrest, pulmonary dysfunction, and CNS side effects such as decreased libido and depression. Similarly, the systemic use another class of drugs, carbonic anhydrase inhibitors, is associated with abnormal production of blood cells, kidney stones, and mental changes such as confusion and depression, paresthesias of the extremities and electrolyte disturbances. Recent efforts to formulate effective topical preparations of these compounds, however, have been encouraging. Although it is assumed that systemic side effects of topical carbonic anhydrase inhibitors will be minimized, it appears that the most serious of these complications; namely blood abnormalities which may impair blood clotting, may be an idiosyncratic and not reversible. That is to say, it is not a dose-dependent effect and may occur after using these drugs only once. Efforts to develop effective aqueous suppressants that offer decreased morbidity and mortality in comparison to those currently available will likely rely on the ability to alter the function of specific cellular events which underlie aqueous humor production by the ciliary epithelium. However, the secretory process which results in aqueous humor production is incompletely understood and the identification of precise cellular mechanisms which underlie this process remain to be established. Although we understand little of the precise way in which aqueous humor fluid is produced, we do know that several classes of “conventional” drugs may theoretically be used to lower intraocular pressure by inhibiting aqueous secretion. However, based on our understanding of how these drugs work, we have reason to think that they, too, would have the potential to cause systemic side effects by affecting the heart, lungs and brain. Recently, a novel family of proteins that regulates water permeability in a variety of tissues has been identified, and the DNA sequences of members of this family have been achieved. The water “channel” regulatory protein is involved in the rapid passage of water across the membranes of various tissues such as red blood cells, collecting tubules of the kidney, and bovine lens. We hypothesize that a member of this protein is likewise present in the ciliary epithelium and has a significant role in the production of aqueous humor fluid. Our preliminary experiments supports the presence of a member of this protein family in the ciliary epithelium. By identifying a protein that may be unique to tissues which transport water, there is a reasonable chance that in the long-term, we can lower intraocular pressure by decreasing aqueous humor production by inhibiting the production of this protein in the ciliary epithelia without causing systemic side effects. In order to do this, we first need to identify the complete structure of this protein which is made by the DNA in these cells. Thus, we propose to clone the full length gene sequence of this protein from ciliary epithelial tissues. After the successful cloning of this gene we further hypothesize that we can regulate its function in transporting water molecules across the membrane. We will perform experiments in which we can manipulate the genetic content of ciliary epithelial cells so that they no longer make this protein. The water permeability of the control cells and altered cells will then be measured by monitoring cell volume in an osmotic swelling assay so that the role of the protein in transporting water can be assessed. Identification of the function of the gene product in regulating the water transport in ciliary epithelium will provide a new insight into specific mechanism(s) which underlies the production of aqueous humor, and thus provide a precise target toward which new glaucoma therapy may be directed.

2nd Year

One of the mainstays of glaucoma treatment is the use of drugs that decrease the secretion of aqueous humor fluid from the ciliary epithelium. Unfortunately, many currently available drugs that decrease aqueous humor production, such as adrenergic antagonists, may cause serious systemic side effects such as cardiac arrhythmia and arrest, pulmonary dysfunction, and depression. Efforts to develop effective aqueous suppressants that offer decreased morbidity and mortality in comparison to those currently available will likely rely on the ability to alter the function of specific cellular events which underlie aqueous humor production by the ciliary epithelium. However, the secretory process which results in aqueous humor production is incompletely understood and the identification of precise cellular mechanisms which underlie this process remain to be established. Water slowly crosses cell membranes by diffusion through the lipid bilayer; however, certain cell types exhibit rapid transmembrane passage of water probably through specialized water-selective channels. Recent evidence suggests the presence of a novel family of proteins that regulates water permeability in a variety of tissues, including kidney, red blood cells, and bovine lens. The resemblance of the function (i.e. rapid passage of water across the membrane) of these tissues to that of the ciliary epithelium has stimulated an effort to seek the presence of water channel protein(s) in the ciliary epithelium. Two degenerate oligodeoxynucleotide primers synthesized corresponding to the conserved amino acid sequences of the water channel family were used in the Polymerase Chain Reaction (PCR) to detect any novel members of this family in ciliary epithelium. A cDNA library constructed from bovine ciliary epithelium served as a template for the PCR. PCR products obtained after 30 cycles, were separated by agarose gel electrophoresis, purified from the melted gel, and directly cloned into the pCR-Script SK(+) cloning vector for DNA sequencing. PCR products separated by agarose gel electrophoresis showed a single major DNA band (400-bp) of the predicted size. Fifty-six different clones containing a 400-bp PCR amplified DNA fragment were sequenced. A search of the amino acid sequence (deduced from the nucleotide sequence) of the PCR amplified DNA from one clone against DNA data base (GenBank) showed the highest homology with the known water “channel” protein family members. However, there were no similarities with any reported gene at the nucleotide sequence level. Cloning of the full length gene from the cDNA library of ciliary epithelium using the partial gene as a probe is currently in progress. These results strongly indicate that the gene for a novel member of the “water channel” protein family is present in the bovine ciliary epithelium tissue. We speculate that the identification of a new water channel protein in the ciliary epithelium will provide new insights into specific mechanism(s) which may underlie aqueous production, and thus provide a precise target towards which new glaucoma therapy may be directed.