Figure 5. Functional contributions of RyRs to intracellular Ca²⁺ release in primary cultured rat ONHAs.

Caffeine (50 mM) was used as RyR agonist to quantify the contributions of RyR to stimulus-induced intracellular Ca²⁺ release. Representative cellular responses (A) and traces (B) are shown. Warmer colors indicate higher fluorescence intensity. We observed an average 13.0 ± 0.8% increase of normalized fluorescence intensity over baseline (F/F₀ max; n=38; C). The experimentally determined AUC for RyR stimulation was 19.5 ± 1.5 iRFU (n=38; D). In our experiments, 56 ± 12% of cells responded to caffeine stimulation (n=3 separate experiments, with at least 15 cells per experiment; E). In order to determine the specificity of the response, we pre-incubated cells with the RyR inhibitor, dantrolene sodium (DAN; 20 µM), for 30 min, and experiments performed with DAN co-application. DAN reduced F/F₀ max (3.0 ± 0.7%, n=49, P<0.001; C) and AUC significantly by 14.6 ± 2.0 iRFU (n=49, P<0.001; D). The number of cells responding to caffeine in the presence of DAN differed significantly by 35 ± 15% (P<0.05; E). * P<0.05; *** P<0.001. Scale bar (in A): 10 µm.