Fig. 5
Ca2+-channel inhibitors differentially regulate general calpain activity, activation of calpain-1 and -2. A Calpain activity assay (blue) in wild-type (wt) and rd1 retinal explant cultures, nuclear counterstain with ToPro (red). Untreated (Untr.) wt and rd1 retina were compared to retina treated with BAPTA-AM, L-cis diltiazem, CM4620, SN-6, D-cis diltiazem, TTA-AS, DS5565, and NA-184. Scatter plot showing percent calpain activity positive cells in outer nuclear layer (ONL). Untr. wt: n = 11; Untr. rd1: 23; BAPTA rd1: 10; L-cis rd1: 6; CM4620 rd1: 5; SN-6 rd1: 9; D-cis rd1: 6; TTA-A2 rd1: 7; DS5565 rd1: 6; NA-184 rd1: 8. B Activated calpain-1 (cyan) immunostaining in wt and rd1 retinal explant cultures with DAPI (grey) as nuclear counterstain. Untreated specimens were compared to retina treated with compounds as in A. Scatter plot showing percent ONL cells displaying calpain-1 activation. Untr. wt: n = 7; Untr. rd1: 17; BAPTA rd1: 9; L-cis rd1: 9; CM4620 rd1: 10; SN-6 rd1: 8; D-cis rd1: 10; TTA-A2 rd1: 8; DS5565 rd1: 8; NA-184 rd1: 9. C Activated calpain-2 (yellow) immunostaining in rd1 and wt retinal explant cultures with DAPI (grey) as nuclear counterstain. Untreated specimens were compared to retina treated with compounds as in A. Scatter plot showing percent ONL cells displaying calpain-2 activation. Untr. wt: 10; Untr. rd1: 21; BAPTA rd1: 9; L-cis rd1: 9; CM4620 rd1: 10; SN-6 rd1: 7; D-cis rd1: 10; TTA-A2 rd1: 8; DS5565 rd1: 6; NA-184 rd1: 9. Note the significant elevation of calpain activity/calpain-2 activation caused by CNGC and CRAC inhibition. The opposite is observed for calpain-1 activation. Statistical testing: one-way ANOVA and Tukey’s multiple comparison post hoc test; significance levels: * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; error bars represent SD; INL = inner nuclear layer, GCL = ganglion cell layer; scale bar = 50 µm