Fig. 5. Effects of extracellular cADPR on [Ca²⁺]_i and O₂ consumption in A. polypoides. (A) Fura 2-loaded sponge cells were incubated at 16°C in Ca²⁺/Mg²⁺-free artificial SW in a thermostated microfluorimetric cuvette, under continuous stirring. The arrow indicates addition of cADPR. Trace a: 200 µM cADPR. Trace b: 10 µM cADPR. Trace c: cells pre-incubated with 30 µM 8-Br-cADPR for 30 minutes prior to addition of 10 µM cADPR. The fluorescence emission ratio E340/E380 is shown. (B) O₂ consumption (left panel) and dye filtration (right panel) of A. polypoides fragments (approx. 5 cm length) were recorded for 2 hours before (control) and after addition of cyclase activity (2.5 nmoles cADPR/ml/minute), 100 µM NAD⁺ or 100 µM cADPR (see key). The calculated slope of the linear regression curves (R0.98) relative to control is shown. Data are the mean±s.d. of four experiments.