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
* Author for correspondence (e-mail: ezocchi{at}unige.it)
Accepted 18 November 2002
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Summary |
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Short-term stimulation followed by long-term depression of amino acid incorporation, oxygen consumption and water filtration were observed after exposure of the sponge to a brief heat stress or to micromolar ABA. These effects could be prevented by the targeted interruption of the signaling pathway either at the level of the cation channel thermosensor or at the level of the cADPR-induced intracellular calcium increase. Moreover, release of cyclase activity into the sea water and generation of extracellular cADPR were observed following brief heat stress. Intact sponge cells were sensitive to extracellular cADPR and addition of purified cyclase increased sponge respiration similarly to heat stress.
This is the first observation of functional effects exerted on Metazoa by the phytohormone ABA: conservation of the ABA/cADPR stress-signaling cascade points to its early evolution in a common precursor of modern Metazoa and Metaphyta. The functional effects induced by extracellular cyclase/cADPR suggest an evolutionary origin of cADPR as an ancient stress hormone in Porifera.
Key words: Abscisic acid, cADPR, [Ca2+]i, Respiration, Marine sponges, Heat stress
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Introduction |
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Recently, cADPR has been shown to mediate temperature signaling in sponges
(Zocchi et al., 2001a), which
are members of the phylum Porifera and comprise the phylogenetically oldest
multicellular animals (Metazoa) (Rodrigo
et al., 1994
). In Axinella polypoides (Demospongiae,
Axinellidae), ADP-ribosyl cyclase, the enzyme responsible for cADPR synthesis
from NAD+, is activated by a temperature increase by an abscisic
acid (ABA)-induced, protein kinase A (PKA)-dependent mechanism. The
thermosensor triggering this signaling cascade is a heat-activated cation
channel. Elucidation of the complete thermosensing pathway in A.
polypoides highlights several features conserved in higher organisms: the
cation channel thermosensor shares all the functional characteristics of the
mammalian heat-activated background K+ channel responsible for
central and peripheral thermosensing, including sensitivity to mechanical
stress and anesthetics (Maingret et al.,
2000a
; Maingret et al.,
2000b
; Kindler et al.,
1999
); involvement of the phytohormone ABA and of cADPR as its
second messenger is reminiscent of the drought-stress signaling cascade in
higher plants (Wu et al.,
1997
). Thus, these observations suggest an ancient evolutionary
origin of an ABA/cADPR-mediated stress-signaling pathway in a common precursor
of modern Metazoa and Metaphyta.
Here, we investigated the functional effects produced on sponge physiology by activation of the temperature-signaling cascade. As the most-simple metazoans, sponges lack a defined tissue organization and display a high degree of cell plasticity. Cells are embedded in a collagenous matrix, impregnated with siliceous or calcareous spicules, surrounding a complex network of internal canals: the main function of sponges is the filtration of water, driven by the synchronized beat of a flagellar epithelium lining the channels. Sponges represent an attractive model system to study the ABA/cADPR interplay because of the following features: (1) the absence of organs and tissues limits the possible functional responses induced by cADPR on these organisms; (2) the absence of tight intercellular junctions enables the preparation of cell suspensions by simple squeezing of the sponge; and (3) basic biochemical mechanisms of signal transduction are present in these lower Metazoa, as exemplified by the above-described signaling pathway. Thus, results obtained with this model system could reveal biochemical pathways conserved in higher organisms.
We investigated the effects of heat stress and ABA on amino acid incorporation and oxygen consumption (as a measure of sponge metabolism) and on the filtration rate (as a measure of sponge functional activity) in A. polypoides. Short-term stimulation followed by long-term depression of these functional activities was observed after exposure of the sponge to heat stress or ABA. These effects could be prevented by the targeted interruption of the temperature-signaling cascade obtained with the cation channel inhibitors bupivacaine and Gd3+, with the cell-permeant cADPR antagonist 8-Br-cADPR and with the intracellular Ca2+ chelator EGTA-AM. Moreover, release of cyclase activity into the sea water (SW) and generation of extracellular cADPR following heat stress, together with the observed effects of extracellular cADPR and cyclase on sponge [Ca2+]i and respiration, point to a hormone-like function of cADPR in Porifera.
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Materials and Methods |
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Intracellular Ca2+ measurements
Intact sponge cells (approximately 8 µm diameter) could be easily
obtained by gentle squeezing of cleanly cut A. polypoides fragments.
Cell viability, as checked microscopically, was always 95% after
mechanical dissociation and following exposure to heat stress or ABA. Loading
of freshly dissociated A. polypoides cells with FURA 2-AM and
Ca2+ measurements were performed as described
(Zocchi et al., 2001a
).
Calibration to obtain the [Ca2+]i from the fluorescence
emission ratio E340/E380 was performed as described
(Zocchi et al., 1998
) except
that cells were permeabilized to Ca2+ with 0.01% Triton. EGTA-AM
(Calbiochem) loading and 8-Br-cADPR (Sigma) treatment of the cells, when
needed, were performed before and immediately after FURA-loading,
respectively: cells were incubated at 16°C for 30 minutes with 20 µM
EGTA-AM, and 10 µM FURA 2-AM (Calbiochem) was then added for a further 90
minutes incubation or FURA 2-loaded cells were incubated with 10 µM
8-Br-cADPR for 30 minutes, then washed in SW and exposed to 24°C in the
cuvette.
[35S]Met/Cys incorporation
For weight determination, A. polypoides tissue fragments were cut
underwater with a sharp scalpel, rapidly blotted on filter paper to remove
excess SW and immediately transferred into a pre-weighed vial containing
SW.
Freshly cut A. polypoides fragments (approximately 350 mg wet weight) were incubated in SW at 24°C for 3 hours in the presence of 20 µM EGTA-AM, or 10 µM 8-Br-cADPR, or without any addition. Duplicate incubations were performed at 16°C (controls). Tissue pieces were then transferred into fresh SW at 16°C and further incubated for 6 or 21 hours. [35S]Met/Cys (Amersham, 2x106 µCi/tube) was added to the tubes for the last 6 hours before harvest. A duplicate series of samples, containing 100 µM cycloheximide (CHX, Sigma), was incubated in parallel to determine the amount of radioactivity incorporation not due to eukaryotic protein synthesis but to possible bacterial contamination. For determination of the incorporated radioactivity, tissue fragments were rinsed in SW, and cells were dissociated mechanically and washed extensively in SW by centrifugation. The residual stroma was repeatedly rinsed in SW by careful squeezing with forceps, and released cells were pooled with the previously dissociated ones. Cells and stroma were washed until the radioactivity detected in the supernatants was negligible. Cells were then lysed with 1 ml deionized water and 300 µl were bleached by the addition of 100 µl hypochlorite. Stroma was blotted dry on filter paper and cut into smaller pieces. Scintillation liquid (Packard) was then added to the cell and stroma samples, and the radioactivity was determined in a ß-counter. Radioactivity incorporation was normalized to a fragment wet weight of 350 mg and subtracted of the radioactivity incorporated in the CHX-treated duplicate sample.
Dissolved O2 measurements
The O2 concentration was continuously monitored with a dissolved
O2 meter (HI9143, Hanna Instruments Italia) equipped with an
incorporated thermometer (±0.1°C precision) and adjustable salinity
setting. The O2 consumption of the electrode was negligible
compared with the sponge respiration. Experiments were performed in natural SW
(salinity 3.9%) and at an ambient temperature of 16°C. Briefly, a cleanly
cut A. polypoides fragment (approx. 3 cm length and 2.5 g wet weight)
was positioned in 150 ml fresh SW in a beaker on a stainless steel tray at
least 24 hours prior to the experiment, in order to let it overcome the
possible stress of handling. Continuous slow stirring was obtained with a
small magnetic bar positioned under the tray. Before measures of O2
consumption, the SW was changed by means of a peristaltic pump. The electrode
was inserted through a rubber cap that ensured air-tight closure of the
beaker. Air bubbles inadvertently trapped below the cap during sealing were
carefully removed with a syringe, inserted through the rubber cap, which also
served to add chemicals during the measurements. A. polypoides
O2 consumption was linear in the range from 9 ppm (the
O2 concentration in non-aerated SW at 16°C) to 3 ppm at a rate
of 1.1±0.34 ppm/h (n=14): however, when the O2
concentration in the respiration chamber approached 5 ppm due to sponge
consumption, the rubber cap was removed, the SW was aerated until the
O2 concentration rose again to approx. 8 ppm (10 minutes), the
beaker was sealed again and the measurement resumed. After measuring the
O2 consumption of a sponge fragment for 1-2 hours, the same
fragment was exposed to ABA or heat stress. ABA was added when the SW was
aerated, before re-sealing. Exposure to heat stress was achieved by
substituting the SW in the beaker with heated SW, by means of a peristaltic
pump. Continuous stirring ensured thorough mixing of the water. When the water
temperature reached 24.0°C, the beaker was transferred at room
temperature. After 30 minutes (final water temperature approx. 23°C), the
beaker was transferred to the cold room, the water temperature was lowered
again to exactly 14°C by mixing with chilled SW, and measurements of
O2 consumption were resumed. O2 consumption during heat
stress was not monitored due to temperature-dependent variations in
O2 solubility, which would have complicated interpretation of the
results. After measuring the O2 consumption for approximately 10
hours, the cap was removed, half of the SW was changed (ABA was added when
needed), the SW was aerated overnight and measurements were resumed the
following day. Under these conditions, respiration of control, unstimulated
sponges was linear for 48 hours.
Dye filtration
This assay investigates the ability of the sponge to extract nutrients from
the SW by means of its filtering activity. Driven by the beat of a flagellar
epithelium, water circulates in the sponge acquiferous system and
molecules/unicellular organisms are retained by the cells lining the internal
canals. The same experimental set-up as described above for O2
consumption was utilized, except that the beaker (250 ml) was left uncovered
and neutral red (BDH Laboratories, London) was added to the SW at a final
concentration of 8 µg/ml. The optical absorbance (at 455 nm) of SW
aliquots, taken every 30-60 minutes, was measured. No dye removal from the SW
was observed with sponges killed by incubation with 2% formaldehyde for 30
minutes. A. polypoides filtration was linear for at least 8 hours,
with one dye refill. The unicellular algal suspensions
(Nanochloropsis sp.) used in the filtration experiments
(Riisgard et al., 1993)
performed in parallel to the dye were kindly provided by the aquarium of
Genova.
ADP-ribosyl cyclase, NAD+ and cADPR detection in SW
Whole, young A. polypoides animals, approx. 6 cm in height (5 g
wet weight) were acclimatized for 2-3 days in 250 ml beakers, in natural SW
under aeration, which also ensured mixing of the SW. The SW was changed daily
with a peristaltic pump. At the beginning of the experiment, the SW was
changed after 4-6 hours, and the SW was substituted with heated SW until the
temperature reached 24°C. The beaker was then transferred at room
temperature under aeration for 30 minutes. Samples of SW were removed at the
beginning of the experiment (time_zero) immediately before and 30 minutes
after the onset of stress. ADP-ribosyl cyclase activity was assayed at
16°C on 200 µl SW aliquots by addition of 2 mM NAD+
substrate. The cADPR produced was detected by HPLC
(Zocchi et al., 2001a).
Detection of ADP-ribosyl cyclase in 50-fold concentrated (Microcon, Millipore)
SW was performed on mildly denaturing SDS-PAGE, as described
(Zocchi et al., 2001a
).
NAD+ and cADPR in the SW were detected by a microfluorimetric
cycling assay (Graeff and Lee,
2002
), performed on 500 µl SW aliquots. Preliminarily, we
verified that the assay could be performed in SW. The enzyme treatment for the
degradation of endogenous NAD+ proved to be completely effective
also in SW. The cADPR standard curve in SW was linear and the sensitivity of
the assay was comparable with that obtained in phosphate buffer. No cyclase
activity, NAD+ or cADPR were detected in the SW at time_zero.
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Results |
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Heat stress had been previously shown to induce ABA synthesis in A.
polypoides, with ABA in turn stimulating ADP-ribosyl cyclase through PKA
(Zocchi et al., 2001a).
Indeed, the concentration of ABA in sponge tissue increased rapidly following
exposure of A. polypoides fragments to 24°C from a basal value of
5.8 pmoles/g to 25, 140 and 180 pmoles/g after 2 minutes, 60 minutes and 4
hours, respectively (results from a representative experiment), with kinetics
similar to those already described (Zocchi
et al., 2001a
). Thus, we also investigated the short- and
long-term effects of ABA exposure on [Ca2+]i. Indeed,
addition of 50 nM ABA at 16°C resulted in an increase in
[Ca2+]i similar in extent and kinetics to that
registered after heat stress (Fig.
1, inset).
The presence of short- and long-term effects of heat stress and ABA on [Ca2+]i in A. polypoides cells prompted us to investigate their functional consequences on sponge physiology at different times after stress induction: during the initial sigmoidal [Ca2+]i increase (i.e. 4-8 hours after heat stress) and after 24 hours.
[35S]Met/Cys incorporation after heat stress
We explored amino acid incorporation into A. polypoides fragments
over two different 6-hour time spans: immediately after heat stress, roughly
overlapping the short-term [Ca2+]i increase, and 24
hours after heat stress, during the `long-term' [Ca2+]i
increase. [35S]Met/Cys incorporation into A. polypoides
cells and stroma increased six- and fourfold, respectively, in the 6 hours
immediately following heat stress, compared with controls kept at 16°C.
Conversely, a marked decrease in amino acid incorporation into cells and
stroma compared with controls was observed 24 hours after heat stress. Both
effects were prevented by pre-treatment of the sponge fragments with EGTA-AM
or with 8-Br-cADPR (Fig. 2).
Thus, a brief temperature increase induces a short-term stimulation, followed
by a long-term inhibition of protein synthesis over the subsequent 6 and 24
hours, respectively. Both effects are mediated by a cADPR-induced increase in
[Ca2+]i. Incubation of sponge fragments at 16°C with
10 µM 8-Br-cADPR alone for 24 hours resulted in a 36% decrease of amino
acid incorporation compared with untreated controls (n=3). This
effect suggests that the antagonist might interfere with a stimulatory effect
of endogenous cADPR on basal sponge protein synthesis.
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O2 consumption and dye filtration after exposure to heat
stress or ABA
Preliminarily, both O2 consumption and dye filtration were
explored in control, unstimulated A. polypoides fragments.
O2 consumption was found to be linear over a range between 9 ppm
and 3 ppm, and could be completely inhibited with 10 mM NaN3,
indicating that it was indeed the result of mitochondrial respiration. When
consumption by the sponge reduced O2 concentration in the SW of the
respiration chamber below 5 ppm, the SW was aerated until the O2
concentration again reached 8 ppm and measurements were resumed. Thus, a
series of parallel lines was obtained over a period of 48 hours (not shown).
Neutral red filtration proved to occur linearly for approximately 8 hours,
with a single dye refill after 4 hours incubation: thereafter, clearance of
the dye decreased, possibly because of sponge saturation. A similar result was
also obtained when an algal suspension was used instead of the dye. In this
case, the decrease of cell number per ml was microscopically determined on
samples of SW removed at different times. We routinely utilized the dye to
investigate effects of heat stress and ABA on sponge filtration: however,
results obtained with the dye were always confirmed with the algal clearance
method. Owing to the above-mentioned saturation, filtration was measured for
not more than 8 consecutive hours, followed by a 12-hour dye- or algal-free
interval before the next measurement was taken
(Fig. 3B,D). Finally, no dye or
algal filtration was detected with sponges killed by incubation in 2%
formaldehyde for 30 minutes.
|
Respiration and filtration proved remarkably comparable between different specimens; nonetheless, each fragment served as its own control, prior to heat stress or ABA exposure. Both functional activities increased immediately after exposure of sponge fragments to heat stress or ABA and comparable effects were produced by the two treatments (Fig. 3): 2.2- and 1.9-fold increases of O2 consumption and 1.6- and 2.0-fold increases in dye filtration were recorded over control values, registered on the same sponge fragment before heat stress and ABA treatment, respectively (values from one representative experiment out of five, giving closely comparable results). The initial stimulation of O2 consumption and filtration (2-6 hours after treatment) was followed by a progressive decrease, down to approx. 50% of control values 24 hours after stress induction (Fig. 3A,C), similar to what was observed for amino acid incorporation.
The effect of inhibitors of the temperature-signaling cascade was then
investigated on short-term stimulation and long-term inhibition of respiration
and filtration induced by heat stress and ABA. The cation channel thermosensor
inhibitors bupivacaine and Gd3+
(Zocchi et al., 2001a) both
prevented the stimulatory effect of a transient heat stress on respiration
(Fig. 4A) and on filtration
(Fig. 4B). Pre-treatment of
sponge fragments with 20 µM EGTA-AM or with 10 µM 8-Br-cADPR also
completely prevented the temperature-induced effects (not shown) as well as
the ABA-induced stimulation of either functional activity
(Fig. 4). Incubation of sponge
fragments with 10 µM 8-Br-cADPR alone, in the absence of any stress
induction, resulted in a progressive reduction of O2 consumption
compared with untreated controls, with a 20% and 30% decrease being recorded
after 2 and 4 hours incubation, respectively. This result, together with a
similar extent of inhibition by the cADPR antagonist on amino acid
incorporation, suggests involvement of endogenous cADPR in the regulation of
basal O2 consumption and protein synthesis in A.
polypoides cells. Finally, incubation of sponge fragments for 2 hours
with 20 µM EGTA-AM or with 10 µM 8-Br-cADPR prior to exposure to heat
stress or ABA completely prevented the long-term decrease of O2
consumption observed 24 hours after stress induction.
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The effect on O2 consumption of 8-Br-cAMP, a membrane-permeant
activator of PKA, was also investigated. Previously, we had demonstrated that
ADP-ribosyl cyclase activation following heat stress or ABA occurs by
PKA-mediated phosphorylation (Zocchi et
al., 2001a). Incubation of A. polypoides fragments with
200 µM 8-Br-cAMP, a membrane-permeant PKA activator, at a constant
temperature of 16°C induced a 30% increase of O2 consumption
(Fig. 4A) and a 50% increase of
dye filtration (Fig. 4B) over
controls during the first 60 minutes after addition.
Effects of extracellular cADPR on [Ca2+]i and
O2 consumption
ADP-ribosyl cyclase activity was detected in the SW (250 ml) surrounding
control, unstimulated A. polypoides sponges. Its levels increased
approx. 20-fold following a brief heat stress (30 minutes at 24°C), from
0.16±0.02 to 3.1±0.25 nmoles cADPR/ml/minute (mean values from
three experiments). A parallel increase of the cADPR concentration in the SW
was observed, from 0.2±0.06 nM to 4.02±0.6 nM, along with a
decrease of the NAD+ concentration from 20±3 nM to
3.0±0.5 nM (mean values from three experiments). The observed efflux of
NAD+ from intact sponge cells (E.Z., unpublished), similar in
extent and kinetics to that reported in mammalian cell lines
(Zocchi et al., 1999), might
account for the presence of the dinucleotide in the SW. Moreover, absence in
the SW of HPLC-detectable (sensitivity limit, 50 nM) ATP (or of adenylic
nucleotides derived therefrom), despite the fact that its intracellular
concentration in A. polypoides is approximately 30 times higher than
that of NAD+ (12.3±1.2 versus 0.4±0.05 nmoles/mg
protein, as detected by HPLC on TCA extracts of freshly dissociated sponge
cells), rules out cell lysis as a possible explanation for the release of
NAD+ and of cyclase activity in the SW.
Several biochemical characteristics of the released cyclase were
investigated: (1) the ratio between cyclase activity on NAD+ and
hydrolase activity on cADPR was comparable with that observed in the cell
lysate (100) (Zocchi et al.,
2001a
) and similar to that reported for the Aplysia
cyclase (Munshi et al., 1999
);
(2) cyclase activity on NAD+ was approximately 20 times higher than
that on the analog nicotinamide guanine dinucleotide (NGD+), again
similar to the cell lysate and to Aplysia cyclase; (3) the molecular
mass of the released cyclase, as detected on mildly denaturing SDS activity
gel, was approximately 20 kDa, i.e. lower than the phosphorylated cytosolic
enzyme purified by affinity chromatography on antiphosphoserine
antibody-coated agarose (Zocchi et al.,
2001a
). Release of cyclase activity and generation of cADPR in the
SW after heat stress prompted us to investigate a possible effect of
extracellular cADPR on the [Ca2+]i of intact sponge
cells. Permeabilized A. polypoides cells were shown to respond to
cADPR and to ryanodine with an immediate [Ca2+]i
increase, which could be completely prevented by pretreatment of the cells
with 8-Br-cADPR (Zocchi et al.,
2001a
). Upon addition of 10 µM cADPR to intact, FURA 2-loaded,
A. polypoides cells, at a constant temperature of 16°C, a slow
and progressive increase of the E340/E380 fluorescence ratio was observed
(Fig. 5A); increasing the
concentration of cADPR (up to 200 µM) resulted in an increase of the slope
of the Ca2+ rise (Fig.
5A). Pre-incubation of the cells with 30 µM 8-Br-cADPR
(Fig. 5A) or with 30 µM
EGTA-AM for 30 minutes completely prevented the Ca2+ rise.
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Finally, we investigated the effect of the addition of cyclase (purified from Aplysia californica; Sigma) on O2 consumption and on dye filtration of A. polypoides fragments. Addition of 2.5 mU/ml (2.5 nmoles cADPR produced/ml/minute), i.e. a cyclase activity similar to that detected in the SW after heat stress (see above), increased the O2 consumption by 40%. NAD+ (100 µM) supplementation to the cyclase or addition of 100 µM cADPR as such induced a similar 50% increase of the respiration (Fig. 5B, left panel). Dye filtration was also stimulated by the presence of extracellular cyclase activity, with or without NAD+ supplementation, or by the addition of pre-formed cADPR (Fig. 5B, right panel).
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Discussion |
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Temperature- and ABA-induced stimulation of respiration and filtration were
also observed on Chondrosia reniformis (Demospongiae, Hadromerida)
(not shown), a sponge that, from a phylogenetic viewpoint, is very far from
A. polypoides (Alvarez and Crisp,
1994). Thus, results obtained on A. polypoides should be
considered representative of a wide spectrum of Porifera.
This is the first observation of functional effects exerted on Metazoa by
micromolar concentrations of the phytohormone ABA, which is involved in
drought- and temperature-stress signaling in plants
(Leckie et al., 1998;
Xiong et al., 1999
):
conservation of the ABA/cADPR signaling cascade from sponges to higher plants
(Wu et al., 1997
;
Leckie et al., 1998
) suggests
its ancient evolutionary origin in a common precursor of modern Metazoa and
Metaphyta. Indeed, ABA has also been detected in the mammalian brain
(Le Page-Degivry et al.,
1986
), although its functional effects and the conditions
modulating its synthesis are still unknown. The stimulation of sponge
O2 consumption (this report) and of plant root elongation
(Spollen et al., 2000
) induced
by ABA might indicate a role of this hormone, and of cADPR as its second
messenger, in the induction of anabolic processes in response to environmental
stimuli. Interestingly, the cation channel thermosensor in A.
polypoides shares several pharmacological and functional properties with
the mammalian two-pore domain K+ channels involved in pH and
O2 sensing in the mammalian brain
(Maingret et al., 2000a
;
Maingret et al., 2000b
;
Patel et al., 1998
).
The inhibitory effect of the cell-permeant cADPR antagonist 8-Br-cADPR on
basal sponge protein synthesis and on O2 consumption suggests a
role for endogenous cADPR in the regulation of basal cell metabolism as well
as in its modification in relation to environmental conditions. In mammals,
from which Porifera are separated by 500 million years of evolution, cADPR has
been recently shown to stimulate cell proliferation of cyclase-transfected
murine fibroblasts (Zocchi et al.,
1998) and of human hematopoietic stem cells incubated with
exogenously added or with stroma-produced cADPR
(Podestà et al., 2000
;
Zocchi et al., 2001b
).
Moreover, cADPR antagonists have been shown to decrease
[Ca2+]i and to inhibit cell growth in both cell systems.
cADPR-induced stimulation of cell proliferation necessarily implies an
increased energy production by the cells to meet the higher demand. An effect
of cADPR on mammalian cell respiration has not been reported so far, although
cyclase activity has been observed in mitochondria
(Yamada et al., 1997
;
Liang et al., 1999
), its
function still unknown.
The long-term inhibitory effects on sponge physiological functions induced
by heat stress or ABA are caused by the profound derangement of the
intracellular Ca2+ homeostasis started by the initial cADPR-induced
intracellular Ca2+ mobilization, which in turn triggers a
protracted extracellular Ca2+ influx, possibly through CRACs
(Reichling and Levine, 1997).
This seems to be supported by the observed inhibitory effect of micromolar
La3+ (Pizzo et al.,
2001
) and capsaicin on the EGTA-inhibitable Ca2+
influx. Capsaicin is being reported as a specific CRAC inhibitor
(Fischer et al., 2001
).
La3+ is also active on voltage-dependent Ca2+ channels
(Carbone and Swandulla, 1990
);
however, their absence in A. polypoides cells
(Zocchi et al., 2001a
)
suggests inhibition of CRACs. The cADPR antagonist 8-Br-cADPR prevents both
the short-term increase and the long-term inhibition of O2
consumption following heat stress or ABA, underlying the causal role of cADPR
in the induction of both responses. The long-lasting effect of a transient,
although sharp, temperature increase provides a sort of cell `memory' via
[Ca2+]i, which negatively affects sponge physiology long
after the return of temperature to lower values. This mechanism might be
responsible for the recently observed mass mortality episode attributed to an
exceptional SW temperature increase in the Northwestern Mediterranean
(Cerrano et al., 2000
). In this
respect, the kinetics of the temperature rise, and of the consequent
cADPR-induced [Ca2+]i increase, might be important: a
sharp, though transient, temperature increase, as investigated here, results
in a sudden increase of [cADPR]i
(Zocchi et al., 2001a
) and of
[Ca2+]i (Fig.
1), which triggers extensive Ca2+ influx and negatively
affects sponge physiology. By contrast, a slow and progressive temperature
increase, as occurs during seasonal cycles, could result in a limited increase
of [cADPR]i and [Ca2+]i, thus stimulating
sponge functional activities.
Finally, an intriguing observation described here is the release of a
low-molecular-weight cyclase (20 kDa) into the SW following heat stress. A
similar effect was registered with the contractile sponge Chondrosia
reniformis following gentle mechanical stimulation (not shown). The
mechanism underlying release of cyclase activity and of NAD+ does
not involve cell lysis, as ATP (or adenylic nucleotides derived therefrom) was
undetectable in the SW despite its intracellular concentration being 20 times
higher than that of NAD+. Cyclase activity has been shown to be
compartmentalized in large granules in Aplysia ovotestis
(Hellmich and Strumwasser,
1991) and granule-rich cells have been observed in A.
polypoides (Van de Vyver and Buscema,
1985
) and in C. reniformis
(Bonasoro et al., 2001
). In
C. reniformis, these cells are frequently observed around the water
canals, where they appear to actively discharge their contents under
conditions of increased sponge plasticity (`creeping')
(Bonasoro et al., 2001
).
Whatever the mechanism involved, release of cyclase activity into the SW
following heat stress suggests a possible role of extracellular cADPR in the
sponge response to environmental stress. Efflux of NAD+ from sponge
cells (E.Z., unpublished), similar to that observed from mammalian cells
(Zocchi et al., 1999
), might
provide the required substrate for the extracellular generation of cADPR.
Indeed, both NAD+ and cADPR were detected in the limited SW volumes
used in these experiments, their levels changing in opposite directions after
heat stress, indicating consumption of the former and production of the latter
by the released cyclase.
Notably, intact sponge cells respond to extracellular cADPR with a
progressive [Ca2+]i increase
(Fig. 5A) and addition of
cyclase activity or of pre-formed cADPR to the SW induces an increase of
sponge O2 consumption (Fig.
5B): these results are reminiscent of the functional effects
(increase of [Ca2+]i and of proliferation) induced by
extracellular cADPR on murine fibroblasts
(Franco et al., 2001) and on
human hemopoietic progenitors
(Podestà et al., 2000
).
In this case, similar effects were obtained with micromolar concentrations of
exogenously added nucleotide or with nanomolar concentrations provided by
ectocyclase-expressing, adjacent stromal cells, highlighting the importance of
close cell contacts for efficient cADPR delivery
(Zocchi et al., 2001b
;
Franco et al., 2001
). These
observations suggest that cADPR might have originated as an ancient stress
hormone, with both intracellular and extracellular functions, and has
maintained these unusual properties in higher organisms, where it can function
both as an intracellular signal molecule and as a cytokine.
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