First published online 8 January 2003
doi: 10.1242/jcs.00277
ABA- and cADPR-mediated effects on respiration and filtration downstream of the temperature-signaling cascade in sponges
Elena Zocchi1,*,
Giovanna Basile1,
Carlo Cerrano2,
Giorgio Bavestrello3,
Marco Giovine1,
Santina Bruzzone1,
Lucrezia Guida1,
Armando Carpaneto4,
Raffaella Magrassi4 and
Cesare Usai4
1 DIMES, Section of Biochemistry, University of Genova, Viale Benedetto XV
no1, 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
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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.
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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 (R 0.98) 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.
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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.
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© The Company of Biologists Ltd 2003
) 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.
) 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 (R