We propose to study the roles of cGMP and cGMP-mediated transmitters in regulating ciliary epithelial function. On a systemic level, the natriuretic peptides (ANP and BNP) and NO on the one hand, and angiotensin II (AII) on the other, form a pair of balancing and opposing transmitters regulating vasomotion; the natriuretic peptides and AII also oppose each other in regulating natriuresis. This integrative balance is partly mediated through changes in the intracellular concentration of cGMP (cyclic guanosine-3',5'-monophosphate), which is increased by natriuretic peptides and NO and decreased by AII. Published data indicate that the ciliary epithelium can respond and/or has receptors to all of these transmitters. Furthermore, on an intracellular level, the two cyclic nucleotides cGMP and cAMP (cyclic adenosine-3',5'-monophosphate) appear to constitute an additional pair of balancing and opposing messengers, regulating the secretion of aqueous humor. On the basis of published data and our unpublished observations, we have formulated a hypothesis concerning the roles of cGMP in regulating the rate of aqueous humor secretion. We propose that the major effect of cGMP is to reverse a cAMP- mediated inhibition of the Na,K-exchange pump, involving both protein kinase A and DARPP-32 of the non-pigmented ciliary epithelial cells. The experimental approach of the current program will be to measure both transepithelial and transmembrane transport by the ciliary epithelium. We shall perform transepithelial measurements and cell-attached patch clamping of the intact ciliary epithelium from both the rabbit (whose characteristics have been well defined) and the cat (whose properties are likely closer to those of primates). We shall also study primary and/or continuous lines of non-pigmented and pigmented ciliary epithelial cells (of human, bovine, rabbit and cat origin) by patch clamping in the conventional whole-cell, perforated whole-cell, cell-attached and excised- patch modes. The specific objectives of the research program are to: (1) determine the effects of each transmitter (ANP, BNP, AII, and NO) separately, and the interactions of these transmitters; (2) test whether the interactions between the transmitters and second messengers (cGMP and cAMP), and between cAMP and cGMP themselves, conform to the hypothesis proposed; and (3) determine whether the cyclic nucleotide-regulated effects are mediated by the transport mechanisms postulated.
