Figure 4. Functional contributions of IP₃Rs to intracellular Ca²⁺ release in primary cultured rat ONHAs.

In order to determine the contribution of IP₃Rs to Ca²⁺ release from intracellular stores, we quantified the rise in intracellular Ca²⁺ concentration upon stimulation with the IP₃R-selective agonist, IP₃, using optical imaging and the Ca²⁺ fluorophore, Fura-2. For delivery, we used the membrane-permeable molecules IP₃-AM (10 µM) and Fura-2-AM (1 µM). Representative cellular responses (A) and traces (B) are shown. Warmer colors indicate higher fluorescence intensity. IP₃ stimulation resulted in a mean 19.0 ± 1.3% increase of normalized fluorescence intensity over baseline (F/F₀ max; n=47; C). This corresponded to an experimentally determined AUC of 168 ± 18 iRFU (n=45; D). In our experiments, 81 ± 13% of cells responded to IP₃ 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 cell with the IP₃R blocker, Xestospongin D (Xes D), for 30 min, and experiments performed with Xes D co-application. Xes D resulted in a 15.4 ± 2.5% reduction of F/F₀ max (n=18, P<0.001; C), and reduced AUC significantly by 145 ± 30 iRFU (n=18, P<0.001; D). The number of cells responding to IP₃ in the presence of Xes D was reduced significantly by 68% (P<0.05; E). * P<0.05; *** P<0.001. Scale bar: 10 µm.