The overall goal of this research continues to be to define the biochemical and functional cross talk between the sympathetic nervous system, which governs the formation of cAMP and muscle relaxation, and the parasympathetic nervous system, which governs the generation of IP3-, DAG-, Ca2+, and muscle contraction. In addition, our studies on the interactions between these systems and the sensory nervous system, which governs the release of neuropeptides, and the local hormones: prostaglandin F2a, endothelin (ET), adrenomedullin (ADM), and atrial natriuretic peptide (ANP) will be continued. Prnviously, we have developed a biochemical correlate for the functional antagonism between these systems. Here, we propose to continue to elucidate the biochemical mechanisms underlying the potential sites of interactions between these signal transduction pathways in the ocular tissues (Sphincter, dilator and ciliary muscles, human ciliary muscle (HCM) cells from normal and glaucomatous eyes, and normal (CISM) and SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. To accomplish these objectives we propose to address the following specific aims:
AIM -I will investigate the cross talk between the cyclic nucleotides (cAMP, cGMP) and the activation of the phospholipase C-beta isoforms (beta1, beta2, beta3), and their physiological consequences (contraction-relaxation and Ca2+ mobilization).
AIM -II will investigate the role of PLC-gamma and tyrosine phosphorylation in the actions of PGF2a, latanoprost, ET and carbachol (CCh) on IP3 production, Ca 2+ mobilization and contraction and their regulation by the cyclic nucleotides.
AIM -III will investigate the role of mitogen-activated protein kinase (MAPK) in the actions of PGF2a, latanoprost, ET, CCh and norepinephrine (NE) on muscle contraction and their regulation by the cyclic nucleotides.
AIM -IV will investigate the molecular mechanisms underlying the effects of ANP, ET, CGRP and ADM on the guanylyl cyclase system and their interactions with the IP3- Ca2+ system in CISM-, SV-CISM-2-, and HCM cells. For many years cyclic nucleotides have been implicated in playing a mediatory role in the IOP-lowering effects of a wide variety of drugs. The mechanisms of the ocular hypotensive effects of these drugs is unclear. Cross talk between the signal transduction pathways represent an important focal point for pharmacological manipulation, the proposed studies will provide major insights into the signal transduction mechanisms mediating the actions of PGF2a, latanoprost, ET, CCh, and NE. Understanding the cross talk between these systems will yield novel information about: (a) Characteristics and functions of ocular receptors; (b) functions and mechanisms of stimulation of PLC-beta, PLC-gamma, MAP kinases and the guanylyl-and adenylyl cyclases; (c) the G proteins that activate the effector enzymes; (d) mechanistic insights into the role of tyrosine phosphorylation in smooth muscle contraction; (d) how does cAMP and cGMP lower intracellular Ca2+ and relaxes smooth muscle; and (e) the mechanism of action of latanoprost and other ophthalmic drugs which will lead to the development of more effective antiglaucoma drugs.
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