Mechanisms of Action of Drugs Which Alter Aqueous Humour Formation

There is a great need for an in vitro model for studying aqueous humour dynamics. A large number of antiglaucoma drugs is currently in clinical use or are in various stages of development, and in some cases their underlying biochemical mechanisms of action are unclear. The present work was undertaken to find out whether the easily obtainable inexpensive bovine eye could be established as such a model to study the physiology and pharmacology of ocular hypotensive drugs. The specific objective was to try to explore the mechanism of action of the extensively used beta-blocker, timolol, to determine whether it acts through the conventional beta-adrenoceptor-adenylate cyclase mechanism, as is often assumed. The isolated arterially perfused bovine eye has been proved to be a valid model for studying aqueous humour dynamics and the pharmacology of various antiglaucoma drugs. Its usefulness has been shown by significant effects of standard and well-established ocular hypotensive agents, such as timolol and MK-927, a carbonic anhydrase inhibitor, on both intraocular pressure and aqueous humour formation. MK-927 produced a dose-dependent decrease in intraocular pressure and aqueous humour formation with moderate doses of 1, 10 or 100 nmol. In addition to the responses to the standard drugs, the viability and metabolic activity of the isolated perfused eye has been shown by significant consumption of O[2] and elaboration of CO[2] during the course of perfusion. This was achieved by comparing the two parameters between the perfused living and dead eyes. The bovine perfused eye has been shown to offer the opportunity to study the effect of ocular hypotensive drugs upon intracellular mechanisms, especially upon second messenger pathways. It allows rapid and convenient sampling of substantial amounts of target tissue for biochemical analysis after drug challenge. The usefulness of the perfused eye has also been shown for isolating and culturing ciliary epithelial cells. Two new methods of isolating and culturing ciliary epithelial cells have been developed. One by aseptic and consecutive perfusion of the isolated eye with DMEM, calcium-free buffer and collagenase and the other by incubating the excised ciliary processes with collagenase. The cells were successfully propagated up to 10 passages without any obvious sign of change of growth characteristics. In first passage cells cultured in this way, there occurred highly significant and concentration-dependent increases in cyclic AMP and cyclic GMP in response to terbutaline and atriopeptin, respectively, indicating that these cells retain functional receptors. This will open up broad opportunities for physiological, pharmacological and biochemical studies on specific target cells responsible for aqueous humour production. (Abstract shortened by ProQuest.)