PURPOSE: To ascertain the expression pattern of alpha(2)-adrenergic receptors in the ciliary body (CB) and determine the effect of brimonidine on matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ciliary body smooth muscle (CBSM) cells. METHODS: Qualitative RT-PCR was performed to detect the mRNA of the alpha(2)-adrenergic receptor subtypes alpha(2)A, alpha(2)B, and alpha(2)C in CB and CBSM cultures. Immunohistochemistry and immunoblot analysis were performed to further investigate alpha(2)A receptor expression in CB tissue and CBSM cells. CBSM cells from 15 different human donors received control or brimonidine tartrate (45 nM) for 1, 3, or 7 days. Changes in pro-MMP-1, -2, -3, -9, and -24 and TIMP-1, -2, -3, and -4 levels were evaluated by Western blot, with GAPDH as the endogenous control. Zymography was used to assess the activity of MMP-1, -2, -3, and -9. RESULTS: The mRNA of alpha(2)A, alpha(2)B, and alpha(2)C were detected in CB tissue and CBSM cells. Immunohistochemistry localized alpha(2)A receptors within the CB stroma. Immunoblot analysis demonstrated production by CBSM cells. Brimonidine increased pro-MMP-9 an average of 116% +/- 34% (P = 0.0360); enzymatic activity of MMP-9 was unchanged. TIMP-4 decreased an average of 25% +/- 8% (P = 0.0329) in conditioned medium, but increased 70% +/- 13% (P = 0.0057) in cell lysates. CONCLUSIONS: The presence of alpha(2)A, alpha(2)B, and alpha(2)C in CB tissue and CBSM cells indicates the possibility that brimonidine affects uveoscleral outflow. However, the changes in MMP-9 and TIMP-4 without significant changes in MMP-9 activity suggest that a role of the MMP/TIMP system in outflow is unlikely.
PURPOSE: To ascertain the expression pattern of alpha(2)-adrenergic receptors in the ciliary body (CB) and determine the effect of brimonidine on matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ciliary body smooth muscle (CBSM) cells. METHODS: Qualitative RT-PCR was performed to detect the mRNA of the alpha(2)-adrenergic receptor subtypes alpha(2)A, alpha(2)B, and alpha(2)C in CB and CBSM cultures. Immunohistochemistry and immunoblot analysis were performed to further investigate alpha(2)A receptor expression in CB tissue and CBSM cells. CBSM cells from 15 different human donors received control or brimonidine tartrate (45 nM) for 1, 3, or 7 days. Changes in pro-MMP-1, -2, -3, -9, and -24 and TIMP-1, -2, -3, and -4 levels were evaluated by Western blot, with GAPDH as the endogenous control. Zymography was used to assess the activity of MMP-1, -2, -3, and -9. RESULTS: The mRNA of alpha(2)A, alpha(2)B, and alpha(2)C were detected in CB tissue and CBSM cells. Immunohistochemistry localized alpha(2)A receptors within the CB stroma. Immunoblot analysis demonstrated production by CBSM cells. Brimonidine increased pro-MMP-9 an average of 116% +/- 34% (P = 0.0360); enzymatic activity of MMP-9 was unchanged. TIMP-4 decreased an average of 25% +/- 8% (P = 0.0329) in conditioned medium, but increased 70% +/- 13% (P = 0.0057) in cell lysates. CONCLUSIONS: The presence of alpha(2)A, alpha(2)B, and alpha(2)C in CB tissue and CBSM cells indicates the possibility that brimonidine affects uveoscleral outflow. However, the changes in MMP-9 and TIMP-4 without significant changes in MMP-9 activity suggest that a role of the MMP/TIMP system in outflow is unlikely.