Wide long lasting perinuclear Ca2+ release events generated by an interaction between ryanodine and IP3 receptors in canine Purkinje cells

The purpose of this study was to determine whether IP3Rs contribute to the generation of wide long lasting perinuclear Ca2+ release events in canine Purkinje cells. Spontaneous Ca2+ release events (elevations of basal [Ca2+] equivalent to F/F0 3.4SD over F0) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca2+ waves were analyzed. Subsarcolemmal region (SSL) was defined as 5 μm from cell edges. Core was the remaining cell. The majority of events (94%, 0.0035 ± 0.0007 events (ev)/μm2/s, N = 34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022 ± 0.00005 ev/μm2/s, N = 41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0 ± 3.9 vs. TE 9.0 ± 0.3 μm2, P < 0.01) and higher amplitude (F/F0 1.38 ± 0.02 vs. TE 1.20 ± 0.003, P < 0.01). WLE event rate was increased by phenylephrine (10 μM, P < 0.01), inhibited by 2APB and U73122 (P < 0.05), and abolished by tetracaine (1 mM) and ryanodine (100 μM). While SSL WLEs were scattered randomly, Core WLEs (n = 69 events) were predominantly distributed longitudinally 18.2 ± 1.6 μm from the center of nuclei. Immunocytochemistry showed that IP3R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs. In Purkinje cells, wide long lasting Ca2+ release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP3R1s evoked Ca2+ release and may play a role in Ca2+ dependent nuclear processes.