Characterization of the Bradykinn Receptor in Human Corneal Epithelium

Wiernas, Terry Kirkham, Characterization of the Bradykinin Receptor in Human Corneal Epithelium. Doctor of Philosophy (Biomedical Sciences), August, 1997, 255 pp., 5 tables, 39 figures, references, 137 titles. Bradykinin (BK) is a well-established mediator of inflammation. High levels of BK in human tears following ocular allergic provocation led to the hypothesis that BK receptors may exist on the corneal epithelium and could play a role in corneal inflammation and/or wound healing, in addition to other functions. To test this hypothesis, human corneal epithelial cells were cultured and used to conduct a series of studies to evaluate and characterize the BK receptor. Due to the limited supply and high cost of primary human corneal epithelial (P-CEPI) cells, in addition to the fact that these cells do not divide and proliferate over more than a few passages, SV40 virus-immortalized human CEPI cells (CEPI-17-CL4) were used as a model system. Extensive studies confirmed that the immortalized cells faithfully represented the primary cells. This study demonstrated the presence of BK receptors on corneal epithelial cells for the first time. The receptors were characterized as the B2 subtype and were found to be represented by an apparent single binding site. Furthermore, stimulation of these receptors was found to elicit concentration-dependent increases in both inositol phosphates, via activation of phospholipase C, and intracellular calcium mobilization. The rank order affinity of BK and its analogs as determined by binding assays was found to correlate well with the rank order potency of BK and its analogs in evoking the latter functional responses, which were blocked by two B2-receptor selective antagonists. A significant, concentration-dependent stimulation of [3H]thymidine uptake in CEPI cell DNA was elicited by BK which suggests a potential mitogenic effect of BK and a role in corneal wound healing. BK did not significantly affect the release of three pro-inflammatory cytokines, prostaglandin E2 or matrix metalloproteinase-1, and seemed to have an inhibitory effect on the release of tumor necrosis factor α. In conclusion, these studies have confirmed that CEPI-17-CL4 cells represent a good in vitro model of human corneal epithelium and have contributed to a better understanding of the ocular effects of BK and characterization of its receptor within the cornea.