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D. J. Rhee, Y. Ooi, D.-J. Oh; Analysis of Alpha-2 Adrenergic Receptors and Effect of Brimonidine on Ciliary Body Smooth Muscle Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1601. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To ascertain the expression pattern of alpha-2 adrenergic receptors in human ciliary body (HCB) stroma and to determine the effect of brimonidine on matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) on human ciliary body smooth muscle (CBSM) cells.
Total RNA was isolated and qualitative RT-PCR was performed to detect the mRNA of alpha-2 adrenergic receptor subtypes α2A, α2B, and α2C in HCB tissue and CBSM cultures. Immunohistochemistry of ciliary process and immunoblot were performed to further confirm the presence of α2A receptor in HCB tissue.Confluent human CBSM cells from 13 different donors were exposed to vehicle control or brimonidine tartrate for 24 hours. Changes of MMP-1, -2, -3, -9, -24 and TIMP-1, -2, -3, -4 levels were evaluated by Western blot and zymography. GAPDH served as an endogenous control.
The mRNA of α2A, α2B, and α2C were detected in HCB tissue and CBSM cells. Immunohistochemistry and immunoblot showed presence and localization of α2A receptors in the stroma of HCB tissue and CBSM cells, respectively. In response to 45 nM of brimonidine, pro-MMP-9 increased an average of 116% ± 34% (p<0.05);enzymatic activity of MMP-9 was unchanged. TIMP-4 expression decreased an average of 25% ± 8% (p<0.05). In whole cell lysates of human CBSM cells, TIMP-4 expression increased an average of 70% ± 13% (p<0.05) in response to 45 nM of brimonidine.
The presence of α2A, α2B, and α2C in HCB tissue and CBSM cells indicates a possible effect of brimonidine on uveoscleral outflow. The coordinated changes of MMP-9 and TIMP-4 without significant changes of MMP-9 activity suggests unlikely role of the MMP:TIMP system in ECM alteration.
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