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The present experimental observations suggest that some alpha/beta-blockers, alpha1-blocker, alpha2-agonist, and prostaglandin derivative stimulate ECM degradation of ocular surface tissue by modulating the balance between MMPs and TIMPs.

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Our present study suggests that modulations of the alpha-adrenergic receptor, alpha1-blocking and alpha2-stimulation, by antiglaucoma eye drops may cause beneficial effects on NMDA-induced retinal damage in the rat.

To study rhodopsin (Rho) phosphorylation and dephosphorylation in Royal College of Surgeons (RCS) rat retina, speci¢c antibodies toward major Rho phosphorylation sites in vivo, 334Ser or 338Ser, were prepared by immunization of authentic phosphorylated peptides in rabbit. Enzyme-linked immunosorbent assay identi¢ed that the raised antibodies exclusively recognized either the phosphorylated 334Ser or 338Ser site. In immuno£uorescence labeling, both antibodies recognized photoreceptor outer segments in light-adapted retinas from SpragueD awley (SD), Brown^Norway (BN) and RCS rat. During dark adaptation, immunoreactivities toward phosphorylated 338Ser and 334Ser sites were diminished within several hours (0.2^2 h) in SD and BN rat retinas. However, those toward phosphorylated 338Ser and 334Ser sites were diminished within 4 to 7 days in RCS rat retinas. In vitro studies demonstrated decreased levels of both Rho phosphorylation and dephosphorylation reactions in RCS retinas. However, the dephosphorylation reaction was much more greatly a¡ected than the phosphorylation reaction. Extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat. ß

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The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys(37)-Xaa(3)-Cys(41)-Xaa-Cys-Xaa(9)-Cys) and a Cys-->Ser (C/S) mutant (Cys(37)-Xaa(3)-Ser(41)-Xaa-Cys-Xaa(9)-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42 kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with the mutant cell line, the wild-type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly.