The overall goals of this research proposal are: (a) to investigate the role of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its derived second messengers in agonist-induced muscle contraction in the sphincter and dilator smooth muscles of the iris-ciliary body, (b) to seek novel interactions between the cAMP and IP3-DG-CA2+ second messenger systems and the tension responses in these muscles, and (c) to investigate the involvement of PIP2 hydrolysis and its derived second messengers in the mechanism of receptor desensitization in the smooth muscles of the iris. Specifically: (I) To understand the biochemical-functional interactions between IP3-Ca2+-DG and cAMP signalling pathways in the smooth muscles of the iris-ciliary body we will: 1. Investigate the relationships between PG-induced IP3 accumulation, DG production, MLC phosphorylation, cAMP formation and muscle tension in the iris sphincter of different species. 2. Investigate the relationships between the effects of various agonists and antagonists, including adrenergic, muscarinic cholinergic, peptidergic and PGs, on the biochemical and functional responses in the bovine sphincter and dilator muscles. 3. Investigate the interaction between the PIP2 pathway and the adenylate cyclase pathway that is mediated through the activation of protein kinase C. 4. Investigate the effects of cAMP and/or its analogues on agonist- induced IP3 accumulation in iris sphincter membrane fractions. (11) To elucidate the role of PIP2 hydrolysis in the molecular mechanisms underlying receptor desensitization of the smooth muscles of the rabbit and bovine iris-ciliary body we will: (1) Investigate alterations in receptor-induced IP3 accumulation, DG production, MLC phosphorylation, cAMP formation and contraction due to in vitro and/or in vivo cholinergic, adrenergic, peptidergic and PG desensitization. 2. Investigate the possibility that protein kinase C-mediated phosphorylation of G proteins and/or receptors could be involved in the mechanism of desensitization. 3. Investigate the possibility that in vivo cholinergic and adrenergic desensitization of the eye may be reversed by administration of myo- inositol. 4. Characterize, through binding studies, the IP3 receptors in the iris muscle membranes and investigate the properties of IP3-induced Ca2+ fluxes in membrane fractions from normal and desensitized muscle. (III) We will investigate the role of MLC phosphatase in the mechanism of agonist-induced muscle relaxation in the iris sphincter. (IV) We will isolate and characterize the smooth muscle cells of the iris sphincter and dilator and investigate the properties of receptor-mediated PIP2 hydrolysis, Ca2+ mobilization, MLC phosphorylation and cAMP formation. Interaction between the cAMP and IP3-CA2+-DG signalling systems represent an important focal point for pharmacological manipulation and the proposed studies will lead to better understanding of aqueous humor dynamics and to the development of more effective and antiglaucoma drugs. In addition, our proposed studies will yield important information on: (a) Properties and function of muscarinic, alpha1-adrenergic, PG and peptidergic receptors in the anterior segment; (b) mechanisms of receptor desensitization in ocular tissues; (c) mechanisms of action of pharmacological agents in the eye; and (d) mechanistic insights into the role of PIP2 and its derived second messengers in smooth muscle function.
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