Cellular Mechanisms in the Ocular Actions of Endothelin

White, Karen A., Cellular Mechanisms in the Ocular Actions of Endothelin. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 1996, 151 pp., 25 tables, 23 figures, references, 111 titles. Endothelins are a family of regulatory peptides which could have important implications in the regulation of aqueous humor outflow and intraocular pressure (IOP). The objectives of this dissertation were to investigate the cellular mechanism of endothelin (ET) receptor interactions in ocular tissues focusing on their effect on second messengers such as phospholipase C (PLC) and calcium, and their interactions with phospholipase A2 (PLA2) in ciliary muscle cells. The hypothesis was that in human ciliary muscle (HCM) cells, endothelin-1 (ET-1), via the ETA receptor and a pertussis toxin sensitive G-protein, activates PLC, which in turn stimulates calcium mobilization. Independent of this pathway, ET-1 also activates PLA2 and increases the release of prostaglandins. These two pathways provide a cellular second messenger balance that influences ciliary smooth muscle contraction. The current study demonstrated that ET-1 and endothelin-2 (ET-2) stimulate calcium mobilization in HCM cells via an ETA receptor subtype. It appears that the increase in intracellular calcium ([CA2+]) is the result of ET coupled to PLC via a pertussis toxin sensitive G-protein. A biphasic calcium response is elicited with ET stimulation consisting of a transient increase in [Ca2+]I which appears to be primarily due to release of intracellular stores, followed by a lower sustained phase which appears to be dependent on the influx of extracellular calcium. Endothelin-1 also appears to stimulate an increase in prostaglandin E2 (PGE2) formation through activation of PLA2. Furthermore, it appears that the effects of ET-1 on PLC and calcium are independent of the ET-1 effects on PGE2 production, such that the ET-1 induced increase in [CA2+]I are coupled to the PLC signaling pathway, whereas increase in PGE2 production appears to be the result of an ETA receptor coupled to PLA2. Whether there are different subtypes of ETA receptors or the receptor is coupled through different G-proteins is uncertain. Endothelin-1 and Big ET-1 immunoreactivity was also observed in both HCM and human nonpigmented ciliary epithelial (HNPE) cells. This is the first time that ET-1 and Big ET-1 immunoreactivity has been detected in the HCM cells, suggesting that these cells have the capability to synthesize both peptides. Furthermore, the increase in ET-1 and Big ET-1 immunoreactivity upon stimulation with TNF-α suggests that cytokines may be important regulators of ET synthesis and release. The findings of this research aid in the understanding of the mechanism of action whereby ETs regulate aqueous humor dynamics and IOP. Through a better understanding of the cellular actions of ET, insight is gained into the development of new ocular selective agents acting at the ET receptor.