This proposal will focus on three intriguing and recently discovered regulatory pathways capable of altering intraocular pressure (IOP). The first concerns ANF and the mechanisms by which this peptide alters secretion in the eye and brain. Experiments described in this grant identify a new signal transduction pathway for ANF, in which this peptide, via a cGMP 2nd messenger, can regulate the phosphorylation of two phosphatase inhibitors - DARPP-32 and Inhibitor-1 - previously thought to be regulated only via cAMP-activated protein kinase. Because of recent compelling evidence concerning the modulation of Na+K+ATPase by dopamine and DARPP in the kidney, this proposal will explore the possibility that ANF may regulate secretion in the ciliary process via a mechanism involving generation of cGMP, G kinase-mediated phosphorylation of DARPP-32 and Inhibitor-1, and inhibition of epithelial cell Na+K+ATPase. In other recent experiments, this Project has identified and characterized a new class of ocular hypotensive agents, the nitrovasodilators (NV's). These agents were a rational development of an effort to bypass the particulate ANF receptor and target the ANF 2nd messenger cascade via a different route - the cytosolic nitric oxide(NO)- sensitive guanylate cyclase (GC). NO-GC is more widely distributed than ANF-activated GC, and NV's have prominent effects on aqueous outflow. Through the use of tissues, cultured cells, and the organ-cultured outflow system, this part of the proposal will investigate the biochemical and physiological mechanisms of action of the NV's in the outflow pathway, and determine the effects of agents known to regulate key pathway enzymes, including NO synthase, soluble-GC, and G-kinase. This pathway has recently taken on new significance with the identification, in brain, of NO as a natural modulator present in neurons and target cells. Accordingly, anatomical localization of NO-synthase and soluble-GC will also be used to determine if an endogenous NO system exists in the anterior segment. The 3rd part of this proposal concerns recent findings on the mechanism of action of epinephrine (EPI) on increasing outflow facility. These findings resulted from our development, with Dr. Erickson-Lamy, of a novel method to simultaneously measure facility and biochemical changes in 2nd messenger production in the organ-cultured human outflow system. The data show that, although cAMP rises almost immediately following EPI. It is proposed to use a combination of biochemical and physiological measurements, as well as selective inhibitors, to systematically determine the site of action of this delay. All three specific aims of this grant have relevance to basic mechanisms of fluid dynamics and to the identification of new pharmacological sites for control of IOP.