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S Ahuja, P Ahuja, AR Caffé, P Ekström, J Wassélius, K Håkansson, M Abrahamson, T van Veen; In vitro Secretion of Proteases and Cystatin C by Retinal Explants of C3H rd/rd and C3H +/+ mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2727.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The retinal explants (RE) are successfully maintained in serum free chemically defined R16 media, to evaluate the effects of various rescue factors. Such media conditioned (CM) by RE need to be analysed for biomolecules influencing the physiopathology of RE and to partially understand the mechanism of action of the rescue factors. Preliminary analysis showed presence of hydrolysed products in the CM. Therefore, the CM was analysed for proteases and cystatin C- an endogenous cysteine protease inhibitor- to study their role in retinal degenerations in rd/rd mice in the presence and absence of glutathione-S-transferase (GST mu). Methods: RE from postnatal day (PN) 2 and PN7 of C3H +/+ and C3H rd/rd mice were cultured upto PN28, in serum free medium with and without GST (10ng/ml). Thrice a week the CM was replaced with fresh medium and analysed for proteases (by fluorometry, gelatin SDS-PAGE) and cystatin C (by ELISA). Results: Compared to PN2, the RE of PN7, +/+ and rd/rd mice secreted more of cystatin C and protease(s) per unit volume of CM. The RE of PN2 and PN7 rd/rd secreted proteases for initially longer period than the comparable RE of +/+ mice. Major proportion of protease(s) from RE of PN7 mice especially the +/+ mice was due to cysteine proteases (irreversibly inhibited by E-64) and those by RE of PN2 were poorly inhibited by E-64. In general GST significantly increased secretion of cystatin C from RE of PN2 rd/rd and PN7 +/+ mice. For short initial periods GST increased secretion of proteases by RE from PN7 and PN2 rd/rd mice. Conclusion: The imbalance in the relative amounts of cystatin C and proteases secreted by the RE of rd/rd mice with and without GST, may modify the normal formation of extracellular matrix and its associated regulatory molecules. Because cysteine proteases require -SH groups for activity, their relative reactivity could also be influenced by the outcome of GST action. These preliminary observations need to be confirmed by analysing larger number of samples.
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