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First published online 8 January 2003
doi: 10.1242/jcs.00277

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ABA- and cADPR-mediated effects on respiration and filtration downstream of the temperature-signaling cascade in sponges

¹ DIMES, Section of Biochemistry, University of Genova, Viale Benedetto XV n^o1, 16132 Genova, Italy
² DIPTERIS, University of Genova, Corso Europa 26, 16132 Genova, Italy
³ Istituto di Scienze del Mare, University of Ancona, Via Brecce Bianche, 60131 Ancona, Italy
⁴ Institute of Cybernetics and Biophysics, National Research Council, Via De Marini 6, 16149 Genova, Italy

View larger version (17K): [in a new window]   Fig. 1. Short- and long-term effects of temperature and ABA on [Ca2+]i in A. polypoides cells. FURA 2-loaded cells were incubated at 24°C in SW without (•) or with 4 mM EGTA () in a thermostated microfluorimetric cuvette, under continuous stirring. EGTA-AM loading () or 8-Br-cADPR treatment () of the cells were performed before and after FURA-loading, respectively. The fluorescence emission ratio E340/E380 was acquired every 30 minutes and calibrated to obtain the corresponding [Ca2+]i; each point is the mean of four values. The double bars indicate interruptions in the x- and y-axis. Inset: trace showing the increase in [Ca2+]i as continuously recorded after addition of 50 nM ABA at a constant temperature of 16°C. Traces are from one of five comparable experiments, performed on different animals and giving similar results.

View larger version (17K): [in a new window]   Fig. 2. [35S]Met/Cys incorporation into A. polypoides tissue fragments after exposure to a temperature rise. Freshly cut A. polypoides fragments were incubated in SW at 24°C for 3 hours with 20 µM EGTA-AM, or with 10 µM 8-Br-cADPR, or without any addition (see key). Tissue pieces were then transferred into fresh SW at 16°C and [35S]Met/Cys was added to the tubes for the last 6 hours before harvest. Radioactivity incorporation, determined as described in Materials and Methods, is expressed as variations relative to respective controls, i.e. fragments incubated at 16°C throughout the experiment. x-axis numbers indicate the total length of the incubations. Results are the mean±s.d. of four experiments, performed on different animals.

View larger version (16K): [in a new window]   Fig. 3. Effects of heat stress and ABA on O2 consumption and dye filtration in A. polypoides. O2 consumption (panels A and C) and dye filtration (panels B and D) were measured at 16°C on A. polypoides fragments (approx. 5 cm length) before (control, ) and after exposure to 24°C for 30 minutes () or addition of 20 µM ABA (). Results shown in each panel are from one representative experiment out of five, performed on different animals. The calculated slope of the linear regression curves (R0.98), relative to the respective control, is shown. The arrows indicate aeration of SW (panels A and C) or dye addition to the SW (panels B and D).

View larger version (18K): [in a new window]   Fig. 4. Effect of inhibitors of the temperature-signaling cascade in A. polypoides on temperature- and ABA-induced short-term stimulation of O2 consumption and filtration rate. O2 consumption and dye filtration were recorded for 2 hours before (control) and after exposure of A. polypoides fragments to heat stress, ABA or 8-Br-cAMP. EGTA-AM (15 µM) and 8-Br-cADPR (10 µM) were added 30 minutes before ABA (20 µM). Bupivacaine (BUPI; 1 mM) and GdCl3 (50 µM) were added during heat stress (24°C for 30 minutes). The calculated slope of the linear regression curve (R0.98) is compared with that of the respective control, i.e. the same fragment prior to stress induction or addition of chemicals. Data are the mean±s.d. of five experiments.

View larger version (22K): [in a new window]   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.