Eicosanoid production and glucocorticoid regulatory mechanisms in cultured human trabecular meshwork cells.
The techniques we developed to propagate HTM cells in serial cell culture have provided an opportunity to investigate the spectrum of endogenous PGs and other eicosanoids that are produced by these cells. PGE2 and PGF2 alpha were the major cyclooxygenase products detected by both radioimmunoassay and thin-layer chromatography. A small amount of 6-keto PGF1 alpha was also detected, indicating that these cells are able to produce prostacyclin. The observation of a substantial increase in the proportion of PGF2 alpha relative to PGE2 at later time periods after a media change suggests a metabolic conversion of PGE2 to PGF2 alpha by these cells. Bradykinin, thrombin, platelet activating factor, and serum were found to be effective stimulators of PG production by HTM cells, whereas calcium ionophore produced only a minor effect. Using high pressure liquid chromatography, elution profiles of radiolabeled metabolites of AA suggested the presence of certain lipoxygenase products, including LTB4, 12-HETE, 15-HETE, and a small amount of 5-HETE in HTM cells. The formation of these products was inhibited by both DEX and BW 755c, reinforcing the view that metabolic conversions of AA through the lipoxygenase pathway were possible in the trabecular meshwork. We also examined the effects of glucocorticoids on specific protein synthesis in the HTM cells, using 35S-methionine labeling and SDS-PAGE techniques. Short-term (1 day) DEX treatment revealed a major induction of a protein band at approximately 30 kDa. Longer treatments (1 to 3 weeks) resulted in major inductions at approximately 55 kDa inside the cells, with the presence of secreted forms (probably glycoproteins) between 55 and 72 kDa. The short-term DEX effect on protein synthesis a phospholipase inhibitor regulating eicosanoid production within the HTM. The longer-term induction may, on the other hand, be related more directly to the development of steroid glaucoma, based on our findings that the inductions of these proteins correlate with the observed time course and dose-dependence topical glucocorticoid effects on IOP. Continued in vitro and in vivo evaluations of the eicosanoid pathways in cultured HTM cells obtained from normal and glaucomatous human eyes may help to delineate their relationship to IOP regulation and the pathogenesis and treatment of glaucoma. Glucocorticoid-induced proteins may be key participants in the regulation of phospholipase activity and hence may represent a major control mechanism of the AA cascade.(ABSTRACT TRUNCATED AT 400 WORDS)
Polansky
J R
JR
Cellular Pharmacology Laboratory, University of California, San Francisco.
Kurtz
R M
RM
Alvarado
J A
JA
Weinreb
R N
RN
Mitchell
M D
MD
D002478:Cells, Cultured; D003907:Dexamethasone / Q000009:adverse effects; D004537:Eicosanoic Acids / Q000378:metabolism*; D004815:Epidermal Growth Factor; D005901:Glaucoma / Q000139:chemically induced; D005938:Glucocorticoids / Q000378:metabolism*; D006801:Humans; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D011453:Prostaglandins / Q000096:biosynthesis; D013997:Time Factors; D014129:Trabecular Meshwork / Q000378:metabolism*
D004537:Eicosanoic Acids; D005938:Glucocorticoids; D011453:Prostaglandins; D004815:Epidermal Growth Factor; D003907:Dexamethasone; D011451:Prostaglandin-Endoperoxide Synthases