• Yorio, Thomas (PI)

Project Details


DESCRIPTION (Adapted from applicant's abstract):
The overall goals of this proposal is to delineate the processes
involved in the regulation of the synthesis and release of endothelins
(ETs) in ocular tissues and to determine endothelin's target sites and
cellular mechanism of action. The hypothesis to test is that ET is
synthesized and stored in ciliary epithelium and other tissues and is
released by a variety of signals, including cytokines to exert paracrine
effects on the ciliary muscle and trabecular meshwork to enhance aqueous
humor outflow and decrease intraocular pressure. Preliminary data from
the applicant's laboratory indicates that the proinflammatory cytokine
TNF-alpha stimulates the synthesis and release of ET-1 from ciliary
epithelium and ET-1 acts on human ciliary muscle through an ETA receptor
to enhance PLC activity and calcium mobilization. This latter effect
is thought to be responsible for the muscle contraction induced by ET.
Although endothelins' actions on intraocular pressure and contraction
of the vascular smooth muscle are well documented, the mechanisms
responsible for the regulation of ocular ET synthesis, release and
actions of endothelins are not fully understood. The following specific
aims are planned to address these mechanisms: (1) to determine if ET,
its precursors and synthetic enzymes are present in human ciliary
epithelium, ciliary muscle and trabecular meshwork cells and tissues
using immunofluorescent microscopy, radioimmunoassay and Western Blot
analysis; (2) to determine the signals and mechanisms responsible for
the regulation of ET's synthesis and release by investigating the signal
transduction pathways activated by TNF-alpha and autonomic agonists,
including the role of protein kinase C (PKC) isoforms, nitric oxide
(NO), and other messengers on the release of ET; (3) to determine tissue
site receptors and cellular mechanism of action released ETs using RT-
PCR, RNase protection analysis and in situ hybridization histochemistry
under normal cytokine, and adrenergic/cholinergic stimulated conditions,
and characterize the signal transduction pathways associated with ET
receptor activation; and (4) to determine the functional role of
released endothelins by linking the signal transduction mechanisms to
contraction in the ciliary muscle and trabecular meshwork by measuring
single cell contraction, myosin light chain phosphorylation, changes in
outflow facility using the isolated anterior segment perfused human eye.
Changes in ion transport process of the isolated ciliary epithelium will
also be examined. These specific aims are designed to determine the
mechanisms involved in the release and synthesis of ET in ocular tissues
and to identify ET target sites. This study will provide information
on the role ETs may play in intraocular pressure (IOP) homeostasis.
Effective start/end date1/12/9830/11/06