Regulation of intracellular pH in cnidarians: response to
acidosis in Anemonia viridis
1,2, Philippe Ganot1, Denis Allemand1,2 and
Julien Laurent1, Alexander Venn1,2, Eric
Tambutte
1,2
Sylvie Tambutte
1 Centre Scientifique de Monaco, MC-98000, Monaco
en Associe
647 ‘Biosensib’, Centre Scientifique de Monaco - Centre National de la Recherche Scientifique, MC-98000,
2 Laboratoire Europe
Monaco
Keywords
amiloride; buffer; cnidarian; EIPA; Na+/H+
exchanger
Correspondence
A. Venn, Centre Scientifique de Monaco,
Avenue Saint Martin, MC-98000 Monaco
Fax: +377 97774472
Tel: +377 97974910
E-mail: [email protected]
Website: http://www.centrescientifique.mc
(Received 18 September 2013, revised 7
November 2013, accepted 11 November
2013)
doi:10.1111/febs.12614
The regulation of intracellular pH (pHi) is a fundamental aspect of cell
physiology that has received little attention in studies of the phylum Cnidaria,
which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either
intrinsic or extrinsic parameters. Corals and sea anemones face natural
daily changes in internal fluids, where the extracellular pH can range from
8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to
experience future CO2-driven declines in seawater pH, a process known as
ocean acidification. Here, we carried out the first mechanistic investigation
to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we
employed confocal live cell imaging and a pH-sensitive dye to track the
dynamics of pHi after intracellular acidosis induced by acute exposure to
decreases in seawater pH and NH4Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium
sp.) and on symbiont-free endoderm cells. Experiments using inhibitors
and Na+-free seawater indicate a potential role of Na+/H+ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular
acidosis in both cell types. We also measured the buffering capacity of
cells, and obtained values between 20.8 and 43.8 mM per pH unit, which
are comparable to those in other invertebrates. Our findings provide the
first steps towards a better understanding of acid–base regulation in these
basal metazoans, for which information on cell physiology is extremely limited.
Introduction
Coral reefs owe their existence to symbiotic cnidarians,
but, despite their enormous economic and ecological
value, coral reef ecosystems are declining globally,
owing to human-induced pressures at local and global
scales. Many of the root causes of coral reef decline
are manifested as physiological stress of the symbiotic
cnidarians that form the structural and trophic basis
of these habitats, but our grasp of the basic physiolog-
Abbreviations
ASW, artificial seawater; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; F, fluorescence intensity; FSW, filtered seawater; NBS, National Bureau of
Standards; NHE, Na+/H+ plasma membrane exchanger; pHe, extracellular pH; pHi, intracellular pH; r, fluorescence intensity ratio; SNARF1 AM, cell-permeant acetoxymethyl ester acetate of carboxyseminaphthorhodafluor-1; SW, seawater; TA, total alkalinity; bHCO3, HCO3
buffering capacity; bi, intrinsic buffering capacity; btotal, intracellular buffering capacity; k2, 640 nm.
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
683
Regulation of intracellular pH in cnidarians
ical properties of cnidarians remains very rudimentary,
particularly at the cellular level [1]. Therefore, there is
an urgent need to advance our understanding of cnidarian cell biology, in order to improve predictions of
how reef-building corals and allied cnidarians respond
to environmental change [2,3].
Intracellular pH (pHi) regulation is a core element
of cell physiology, about which very little is known in
cnidarians. Although recently published research on
cnidarians has started to decipher the role that pH regulation plays in biomineralization and symbiosis [4,5],
it is surprising that, to date, no information is available on the mechanisms by which cnidarians control
pH within the cell. Because most cellular processes are
pH-sensitive, control of pH between narrow limits is
essential for the proper functioning of cells, and is
achieved via membrane-bound transporters and intracellular buffering [6,7]. In many organisms, decreases
in the pH of extracellular fluids (pHe) driven by respiratory CO2 constitute one important example of the
challenge to which the cellular acid–base regulation of
cells must respond. Elevated extracellular pCO2 can
lead to CO2 diffusion into the cell or limit the degree
to which metabolically generated CO2 can diffuse out
of cells, leading to intracellular acidosis by the hydration of CO2 into H+ and HCO3. Evolutionarily conserved mechanisms that respond to this challenge
include Na+/H+ plasma membrane exchangers
(NHEs), which use a transcellular Na+ gradient to
extrude protons from the cell [8–11]. Whereas NHEs
are well characterized in vertebrates and, to a more
limited extent, in marine invertebrates, NHE activity
has never been characterized in cnidarians.
The regulation of pHi of cnidarian cells is important
to our understanding of the symbiosis that many
cnidarians undergo with photosynthetic dinoflagellate
algae (Symbiodinium). This cnidarian symbiosis, which
is among the most widely studied of marine symbioses,
owing to its ecological importance, presents unique
metabolic challenges to the cnidarian host [12,13].
Indeed, symbiotic cnidarian cells are unique among
animals in showing light-driven increases in pHi,
owing to the photosynthetic activity of their algae
[5,14]. Additionally, photosynthesis by the symbionts
and the night-time respiration of both host and symbiont drive wide variations in pHe of internal fluids of
the principal body cavity, the coelenteron [15]. Endoderm cells facing the coelenteron cavity experience dramatic fluctuations in pHe, and these cells must
presumably possess mechanisms for controlling pH to
cope with this environment. These basic mechanisms
of acid–base defense are not characterized, and are of
interest both for a broader understanding of cnidarian
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J. Laurent et al.
physiology, and for the growing research community
trying to improve our mechanistic understanding of
the acid–base regulation of marine invertebrates that
are faced with a steadily decreasing global seawater
(SW) pH driven by the ocean’s uptake of rising atmospheric CO2 (ocean acidification) [16].
In the current study, we used a model cnidarian, the
snakelocks anemone Anemonia viridis. A. viridis offers
certain advantages that make it a suitable model for
the current study and an emerging model for research
into the cell biology of symbiotic cnidarians [5,17,18].
Owing to the difficulties in maintaining continuous cell
cultures of cnidarian cells, investigations into cnidarian
cell physiology are frequently carried out on isolated
cells that cannot be readily localized to a certain tissue
layer [5,19]. Working with A. viridis circumvents this
problem, as its endoderm cell layer can be separated
from the rest of the tissues, providing a cell suspension
containing symbiont-containing and symbiont-free cells
from a known tissue layer.
The current study focused on the response of cnidarian cells to decreases in pHe in the dark, and the
mechanisms involved in this response. There were
three objectives of the study. The first was to characterize the pHi response to extracellular acidification.
Here, we exposed cells to a pHe that corresponded to
the minimum pHe occurring in darkness in the coelenteron cavity of A. viridis (pHe 7.4) [15]. The second
was to investigate the mechanisms underlying the
response of A. viridis cells to induced intracellular acidosis, by use of the classic NH4Cl prepulse approach,
and to examine pHi recovery in cells in the presence of
inhibitors and in the absence of Na+. The third was
to examine the intracellular buffering capacity (btotal)
of A. viridis cells. All experiments were carried out in
the dark, and we performed our study on both
symbiont-containing and symbiont-free endoderm
cells, using in vivo confocal microscopy and the cellpermeant pH-sensitive probe acetoxymethyl ester acetate of carboxyseminaphthorhodafluor-1 (SNARF1 AM). Our findings contribute fundamental information about pHi regulation in cnidarian cells during
intracellular and extracellular decreases in pH.
Results
Response of pHi to extracellular acidification
Isolated endoderm cells were perfused with SW at low
pH to investigate the response to decreased pHe in the
dark (Fig. 1). Cells perfused with SW at the control
value of pH 8.2 maintained a stable pHi of
7.02 0.02 during the 40-min duration of the experiFEBS Journal 281 (2014) 683–695 ª 2013 FEBS
J. Laurent et al.
A
Regulation of intracellular pH in cnidarians
A
B
B
Fig. 1. pHi (mean SEM, n = 15 cells) in symbiont-free cells (A)
and in symbiont-containing cells (B) isolated from A. viridis
perfused for 5 min with SW at pH 8.2, and then for 35 min with
SW at pH 8.2 (control) or pH 7.4, adjusted with HCl.
ment. This value corresponds to resting dark values
observed previously for A. viridis cells [5]. When symbiont-free cells were perfused with SW that had been
previously adjusted to pH 7.4 with HCl, pHi declined
to a value of 6.64 0.06 during the first 15 min, after
which it recovered back to control values during the
following 20 min (Fig. 1A). Similar patterns were
observed for symbiont-containing cells, which showed
a decline in pHi to 6.70 0.07 and then a recovery to
the initial pHi values (Fig. 1B).
The full carbonate chemistry of HCl-adjusted SW,
including pH values presented on both a total scale
and the National Bureau of Standards (NBS) scale,
used in the perfusion experiments are reported in
Table S1.
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
Fig. 2. pHi (mean SEM, n = 15 cells) in symbiont-free cells (A)
and in symbiont-containing cells (B) isolated from A. viridis
perfused for 5 min with SW at pH 8.2 and then for 35 min with
SW at pH 7.4 (adjusted with HCl) containing amiloride (500 lM) or
EIPA (100 lM). Controls were perfused with SW with no inhibitor
at pH 7.4.
Response of pHi to extracellular acidification in
the presence of inhibitors
The response of pHi to extracellular acidification was
investigated in the presence of the NHE inhibitors
amiloride, and 5-(N-ethyl-N-isopropyl) amiloride
(EIPA) (Fig. 2).
Cells perfused with SW at pH 7.4 containing
100 lM EIPA showed similar declines in pHi to those
seen with inhibitor-free treatments in the previous
experiment. However, recovery of pHi did not occur
in the presence of 100 lM EIPA, and pHi continued to
decline throughout the time course in both symbiontfree cells (Fig. 2A) and symbiont-containing cells
(Fig. 2B).
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Regulation of intracellular pH in cnidarians
The response to 500 lM amiloride was less clear-cut.
Initial declines in pHi occurred, but pHi stabilized
after 15 min (reaching stable values of 6.85 0.08 for
symbiont-free cells and 6.83 0.06 for symbiontcontaining cells).
Final pHi values at 40 min were significantly lower
in both EIPA-treated and amiloride-treated symbiontfree cells and symbiont-containing cells than in cells
receiving
inhibitor-free
treatments
(Welch’s
F2,26.464 = 43.997, P = 0.000; F2,42 = 18.620, P = 0.000).
J. Laurent et al.
pHi response to NH4Cl-induced acidosis
The pHi values of symbiont-containing and symbiontfree cells were experimentally manipulated with the
ammonium prepulse technique [20–22]. Control cells
showed the classic response pattern to NH4Cl exposure: initial alkalization on addition of NH4Cl to the
perfusion medium, then intracellular acidosis following
removal of NH4Cl, and finally a gradual recovery of
pHi to initial values in SW at pH 8.2 (Fig. 3).
A
C
B
D
Fig. 3. The dynamics of pHi (mean SEM, n = 15 cells) in symbiont-free cells (A, C) and symbiont-containing cells (B, D) subjected to
acidosis by a prepulse of NH4Cl (20 mM). In (A) and (B), amiloride (500 lM) and EIPA (100 lM) were added at 15 min. In (C) and (D), the
experiment was conducted with ASW or ASW without Na+.
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J. Laurent et al.
When 500 lM amiloride or 100 lM EIPA was added
to SW following removal of NH4Cl, intracellular acidosis occurred, but the recovery of pHi was inhibited
(Fig. 3A,B). Mean pHi values at the end of the experiment were statistically significantly different between
treatments (amiloride, 500 lM; EIPA, 100 lM) for
symbiont-free cells (F2,29 = 3.910, P = 0.031) (Fig. 3A)
and
for
symbiont-containing
cells
(Welch’s
F2,17.860 = 9.486, P = 0.002) (Fig. 3B).
Experiments were also conducted to investigate
whether the recovery of pHi was Na+-dependent. As
this involved exposing cells to artificial SW (ASW), we
confirmed that we obtained the same response to
NH4Cl-induced acidosis when performing the experiment with SW and ASW (compare controls in
Fig. 3A,B with Fig. 3C,D). Whereas pHi recovered
from acidosis in ASW, recovery of pHi did not occur
in Na+-free ASW, and, at the end of the experiment,
pHi was significantly lower in Na+-free ASW than in
ASW for both symbiont-free cells (t22 = 4.989,
P = 0.000) (Fig. 3C) and symbiont-containing cells
(U = 15, z = 3.293, P = 0.001) (Fig. 3D).
Regulation of intracellular pH in cnidarians
A
B
Determination of btotal
We estimated btotal by titrating the cytoplasmic compartment of endoderm cells in the presence of EIPA
(Fig. 4). The values obtained for pHi upon addition of
decreasing concentrations of NH4Cl are shown in
Fig. 4A. From these values, btotal was calculated, and
is plotted in Fig. 4B. The highest btotal values for cells
were observed over the pHi range 6.9–7.35, with a
maximum btotal value (43.87 mM per pH unit) for symbiont-containing cells at pHi 7.30. btotal was slightly
higher in symbiont-containing cells than in symbiontfree cells (+ 25% at pHi 7.5), except for the highest
pHi values (7.97).
Molecular evidence of the presence of NHEs
To look for preliminary molecular evidence that NHEs
are present in cnidarians, we initially performed BLAST
searches in publicly available symbiotic anemone transcriptomes by using sequences for the Human and Drosophila melanogaster NHEs retrieved from GenBank as
bait. These searches did not reveal any genes coding
for NHEs. However, this was not unexpected, as the
A. viridis transcriptome is only partial [18], and fully
sequenced genomes for this species and other symbiotic
anemones (e.g. Aiptasia pallida) are not available.
Instead, we extended the search to a nonsymbiotic
anemone species, Nematostella vectensis, a symbiotic
coral (Acropora digitifera), and representative marine
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
Fig. 4. The relationship of btotal with pHi in symbiont-containing
and symbiont-free endoderm cells treated with EIPA. (A) The
effect of stepwise reduction in NHþ
concentration on pHi
4
( standard deviation). Three experiments were performed on a
total of 15 A. viridis endoderm cells. (B) btotal (mM per pH unit) of
endoderm cells.
invertebrates (the mollusc Crassostrea gigas and the
echinoderm Strongylocentrotus purpuratus), for which
complete genomes are available.
In the anemone N. vectensis and the symbiotic coral
A. digitifera, we obtained six genes encoding NHE
transporters of solute carrier family 9. Phylogenetic
analysis of putative cnidarian NHE sequences with
functionally characterized NHEs in D. melanogaster
and Homo sapiens grouped our putative cnidarian
NHEs with vacuolar, mitochondrial and plasmalemma
isoforms of NHEs (Figs S1 and S2). Other invertebrate
(mollusc and echinoderm) putative NHEs also corresponded to these groups (Figs S1 and S2).
Discussion
The current study investigated how cnidarian cells
cope with decreases in pHi and pHe. The principal
results of the study show that cnidarian endoderm cells
have the capacity to recover from exposure to sus687
Regulation of intracellular pH in cnidarians
tained reductions in pHe, and that this recovery is
mediated by an Na+-dependent, amiloride-sensitive
and EIPA-sensitive system. Additionally, we characterized the buffering capacity of cnidarian cells, which is
also likely to shape their response to decreased pHe.
In the following discussion, we examine these observations in relation to the wider literature on pH regulation, and their significance for cnidarian biology.
Response to extracellular acidification and to
NH4Cl prepulse experiments
In this study, we observed initial decreases in pHi (acidosis) of A. viridis cells caused by external SW acidification and prepulses of NH4Cl. In the case of SW
acidification, we observed a 15-min intracellular acidosis to pHi 6.6. Many cell types show changes in pHi in
response to changes in pHe, depending on the magnitude and duration of the pHe change [6]. Frequently,
the ratio between the change in pHi and the change in
pHe (ΔpHi/ΔpHe) during external acidification is used
as an index of pHi stability. In our experiments, ΔpHi/
ΔpHe was ~ 50%, which falls within the range of
ΔpHi/ΔpHe values that can be calculated from values
in the literature (30–60%) [23–26]. This result was not
influenced by a change in cellular respiration caused
by exposure to SW acidification treatments (i.e. high
rates of intracellular CO2 production), as respiration
rates were not different in cells receiving SW treatments of pH 8.2 and pH 7.4 (t4 = 0.561, P = 0.605)
(Fig. S3). Rather, as SW CO2 concentrations are elevated in our SW acidification treatments relative to
controls (Table S1), we attribute the observed decrease
in pHi to diffusive entry of CO2 into cells, and a limitation of the rate at which CO2 generated by respiration can exit cells by diffusion.
Acidosis of anemone cells caused by NH4Cl prepulse
followed a predictable pattern observed in many other
organisms. Indeed, the mechanism by which this classic
approach causes acidosis has been well described [6,27].
Briefly, when a cell is exposed to NH4Cl solutions containing NH3 and NHþ
4 , the rapid influx of NH3 leads to
an initial rise in pHi, which is attenuated by the simultaneous, although smaller, influx of NHþ
4 . Although most
þ
remains
as
NH
of the entering NHþ
4
4 , a small fraction
dissociates to form NH3 and H+ (governed by the differences between pKa and pHi). When external NH3/
þ
NHþ
4 is removed, the NH4 that previously entered but
failed to dissociate now dissociates into NH3 and H+.
As a consequence, the pHi is lower than the initial value.
In the case of both of the techniques that we used to
cause acidosis of anemone cells (i.e. acidification of SW
and exposure to NH4Cl), the key observation arising
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J. Laurent et al.
from these experiments was that acidosis in cnidarian
cells was followed by a recovery phase that brought
pHi back to the initial values. This observation demonstrates the capacity of cnidarian cells to regulate
against decreases in pHi and pHe. This behavior [6]
has also been documented in many other cell types,
such as barnacle muscle fibers [28], rabbit osteoclasts
[29], teleost cells [11], and sea urchin larvae cells [22].
Interestingly, the recovery rate in the presence of
extracellular acidification (pHe 7.4) was significantly
slower {~ 0.0168 0.003 pH units min1 [mean
standard error of the mean (SEM)]} than in NH4Cl
prepulse experiments (pHe 8.2) (~ 0.0726 0.0102 pH
units min1) (t10 = 4.975, P = 0.001). One potential
explanation is that pHe in SW acidification experiments remained much lower than in NH4Cl prepulse
experiments (pH 7.4 versus pH 8.2). The removal of
H+ from cells therefore occurred with a less favorable
proton gradient in SW acidification experiments
(difference between pHe and pHi of ~ 0.8) than in
NH4Cl prepulse experiments (difference between pHe
and pHi of ~ 1.6).
Mechanisms underlying the response of
endoderm cells to internal acidosis
Having observed that both symbiont-containing and
symbiont-free cells recover from declines in intracellular pH driven by external acidification and NH4Cl prepulse, we conducted experiments to investigate the
mechanism underlying the response of endoderm cells
to internal acidosis.
Buffering capacity
Cells can protect their cytosol from rapid pH swings
with their inherent btotal [7]. btotal is a measure of the
ability of a cell to withstand the addition or removal
of H+, without a change in pHi. Accordingly, the
stronger btotal is, the smaller are the pHi decreases
associated with pHe decreases. In the current study,
the values of btotal recorded in A. viridis endoderm cells
lie between 20.8 and 43.8 mM per pH unit. These btotal
values are comparable to those typically reported in
other invertebrate cells, in the range of 16–40 mM per
pH unit [30–34]. Overall, there was little difference in
buffering capacity between symbiont-containing cells
and symbiont-free cells, although, for both symbiontfree and symbiont-containing cells, btotal varied with
pHe. We observed the strongest buffering capacities
for the lowest pHi values. This is frequently observed
in most cell types, because the pKa of most ionizable
groups is below physiological pHi values [27,35,36].
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
J. Laurent et al.
NHEs
Although the role of cellular buffering capacity is to
minimize the amplitude of pH changes within the cell,
cells also regulate pHi by using ion exchangers.
Among the effectors of pH regulation that play a
major role in pHi recovery of cells that experience acidosis, some are ubiquitous and belong to the ‘housekeeping’ family of NHEs. NHEs harness the
electrochemical gradient of Na+ maintained by the
Na+/K+-ATPase to energize the transport of protons
[7]. These transporters are sensitive to inhibitors of the
amiloride family and derivatives such as EIPA. Acid
extrusion by the NEH in intact cells is also known
to be blocked by the removal of extracellular Na+
[37–39].
Our results demonstrate that the recovery following
intracellular acidosis of endoderm cells was inhibited
in the presence of EIPA and amiloride. We observed
greater inhibition of pHi recovery with EIPA than
with amiloride, which is in agreement with the fact
that this inhibitor generally shows a 10–100-fold
higher affinity for NHEs [40]. Furthermore, this recovery following the intracellular acidosis induced by the
NH4Cl prepulse was also inhibited by the removal of
extracellular Na+. Taken together, these findings point
to a role of an NHE in the recovery of pHi from acidosis in A. viridis.
These observations of putative NHE activity in a
cnidarian contribute to the limited knowledge of
NHEs in marine invertebrates relative to better-characterized vertebrate models. Despite the higher affinity
of EIPA than of amiloride for NHE, very few studies
on marine invertebrates have used EIPA to investigate
NHE behavior. Exceptions include functional studies
on NHE activity in the mollusc Mytilus galloprovincialis [26]. Another area of uncertainty concerning the
function of NHE in marine invertebrates extends to
their transport stoichiometry. Previous research on
some marine invertebrate groups (notably crustaceans
and echinoderms) has suggested that marine invertebrate NHEs operate with a 2Na+/H+ transport stoichiometry rather than the electroneutral Na+/H+
stoichiometry found in vertebrates [41]. Further
research is needed to determine the transport stoichiometry of cnidarian NHEs.
Turning back to the comparison of recovery rates
mentioned earlier, previous studies have shown that
low pHe can reduce NHE activity relative to higher
pHe, and thus the rate at which pHi recovers from
an acid load [38,39]. Accordingly, in addition to the
influence of the H+ gradient, which could modulate
the pHi recovery rate, lower activity of NHE in SW
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
Regulation of intracellular pH in cnidarians
acidification experiments (pHe 7.4) than with with
NH4Cl (pHe 8.2) might explain the slower recovery
rate.
Molecular evidence of the presence of NHEs in
cnidarians
To date, there are no published papers on NHEs in
cnidarians, although this family of transporters has
been well characterized in a wide variety of other animal cells [42–47]. The phylogentic analysis presented
here indicates that cnidarians possess vacuolar, mitochondrial and plasmalemma NHE homologs. The
putative anemone and coral NHEs (N. vectensis
NHE1 and A. digitifera NHE1), which are homologous with plasmalemma-resident NHEs (NHE1–
NHE5), are the most interesting in the context of the
present study. NHEs present in the membranes of
most cells are known to be the main isoforms for pHi
homeostasis [7], and are involved in many physiological processes [10,48,49]. Importantly, these NHE isoforms are known to be sensitive to amiloride and
EIPA [10]. Furthermore, putative cnidarian NHEs
share certain amino acid residues with human NHE1
that have been functionally linked to amiloride and
EIPA binding, and Na+/H+ transport by mutational
studies [49] (see alignment in Fig. S2).
The presence of putative plasmalemma-resident
NHE genes in cnidarians is consistent with the physiological data that we have obtained indicating that an
NHE is involved in pHi recovery from acidosis in
A. viridis. Further sequencing of the A. viridis transcriptome and genome is required to identify the NHE
in this species, but the current findings strongly warrant future studies into NHEs in cnidarians, including
work to characterize gene and protein expression
responses to acidosis.
Significance of results to cnidarian pH regulation
In the context of cnidarian biology, although our
study was conducted with acute decreases in SW pHe
that do not directly mimic the more gradual changes
in pHe that occur under physiological conditions [15],
our results provide information that may be of relevance to an understanding of how cnidarian endoderm
cells face diurnal night-time acidification [15].
Importantly, we observed that pHi of anemone
endoderm cells was able to recover quickly from acute
decreases in pHe. This observation contrasts with
many other organisms, in which pHe decreases usually
lead to a sustained reduction in pHi with no recovery
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Regulation of intracellular pH in cnidarians
[36,50–52]. This high capacity of anemone endoderm
cells to regulate pHi in the face of large external
decreases in pH may reflect the natural variation in
pH that they experience diurnally in nature (that is,
the coelenteron pH decreases by 0.8 below that in SW,
owing to respiration by both hosts and symbionts).
This contrasts with mammalian cells, which, by comparison, usually experience a very stable environment
in terms of pH (that is, blood pH tends to vary by
< 0.1) [53].
In the current study, we considered that both buffering capacity and transporters such as NHEs are important for maintaining pHi constant in anemone
endoderm cells during night-time acidification of the
coelenteron. In the case of buffering capacity, btotal
increases in endoderm cells when pHi decreases, and this
process might help cells to avoid large pHi decreases
associated with acidification of the coelenteron. btotal is
the sum of the individual buffering powers of all cytosolic buffers, and is divided into HCO3 (bHCO3) and
intrinsic (bi) buffering capacities, such that
btotal = bHCO3 + bi [6,36]. bHCO3 depends on intracellular CO2 and HCO3 concentrations, and bi is determined by the cellular concentrations of metabolites that
act as buffers, such as PO43-containing molecules and
ionizable amino acid side chains [50]. The presence of
symbionts might be expected to change internal CO2
and metabolite concentrations in cnidarian host cells
that could change bHCO3 and bi, and therefore btotal.
Actually, our results show that btotal is only slightly
higher in symbiont-containing than in symbiont-free
cells, and our patterns of pHi response to SW acidification were similar for symbiont-free and symbiont-containing cells, indicating that the presence of symbionts
has little effect on the response to acidification. This
suggests that btotal conferred by the presence of the symbiont is probably much lower than btotal associated only
with the host cell. This is probably because our experiments were performed in dark conditions, when there is
no transfer of photosynthate from the symbiont to the
host that could have increased btotal.
Our results point to the role of an NHE in the
recovery of pHi from acidosis in A. viridis. These findings suggest that NHE could play an important role in
pH regulation in cnidarians. Indeed, NHEs would be
expected to be involved in the responses of coral and
other cnidarian cells to decreases in SW pH and
increases in SW pCO2 associated with ocean acidification. This subject will constitute an important area of
future research. In a previous study performed on
intact coral Stylophora pistillata, where we investigated
pHi in calcifying cells of the calicoblastic epithelium
exposed to SW acidification, we observed no signifi690
J. Laurent et al.
cant changes in pHi in corals exposed to SW with a
pH of 7.4 (although a significant decrease was found
at pHe 7.2) [54]. Comparisons between this previous
study and the current study are difficult, because the
previous study involved observations after 1 day and
1 year, and not the short-term reductions and recovery
of pHi observed here.
Future work should also include characterization of
the roles of other potential acid-extruding mechanisms
and the role of pH-sensing enzymes in regulating the
activity of cnidarian membrane transporters. Work in
this area has already begun with the recent characterization of bicarbonate-stimulated soluble adenylyl
cyclase in corals [55].
In summary, the current study investigated mechanisms involved in the response of pHi regulation of
cnidarian cells to acidosis. Our findings on pHi regulation constitute an important step towards a better
understanding of the acid–base regulatory abilities of
cnidarians, which is imperative for a better grasp of
the physiology of cnidarians in an era of environmental change.
Experimental procedures
Anemone culture and preparation of cells
Several A. viridis anemones sampled from the Mediterranean sea (Fontvielle, Monaco) were maintained at the Centre Scientifique de Monaco in aquaria supplied with
flowing Mediterranean SW (salinity: 38.2) with a 2% h1
exchange rate, at 19 2 °C. The irradiance level was
100 lmol photons m2s1 photosynthetically active radiation on a 12-h light/dark cycle. Anemones were fed twice a
week with live Artemia salina nauplii.
Cell suspensions were prepared according to Venn et al.
[5]. Briefly, cells were isolated from anemones before each
experiment by scraping endoderm cells from longitudinally
sectioned tentacles into 50 mL of filtered SW (FSW) [5].
The resulting cell suspension, containing a heterologous
population of symbiont-free and symbiont-containing cells,
was filtered through a 0.45-lm Millipore membrane, and
centrifuged once (350 g, 4 min); the pellet of cells was then
resuspended in FSW. Cell preparations were adjusted
with FSW to a density of 3.6 9 105 cellsmL1 for all
experiments.
Preparation of solutions
SW solutions were adjusted to pH 7.4 (NBS scale) by the
addition of HCl. In both cases, the pH was measured with
a pH electrode calibrated on the NBS scale (Seven Easy;
Mettler Toledo, Columbus, OH, USA) and with the indicator dye m-cresol purple (Acros 199250050, NJ, USA)
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
J. Laurent et al.
according to Dickson et al. [56] to determine pH on a total
scale. In the latter case, the absorbance was measured with
a spectrophotometer (UVmc2; Safas, Monte-Carlo,
Monaco). For ease of reference, pH values are consistently
reported on the NBS scale in figures and throughout the
article. Corresponding total scale pH values for SW solutions are reported in Table S1.
SW solutions were also analyzed for total alkalinity
(TA), which was determined via titration of 4-mL samples
with 0.03 M HCl containing 40.7 g of NaCl l1 with a
Metrohm 888 Titrando Dosimat controlled by TIAMO software. TA was calculated by use of a regression routine
based on Department of Energy guidelines [57]. For each
sample run, certified SW reference material supplied by the
laboratory of A. G. Dickson (Scripps Institution of Oceanography, La Jolla, CA, USA) was used to verify acid normality.
Parameters of the carbonate chemistry of SW solutions
were calculated from total scale pH, TA, temperature and
salinity with the free-access CO2SYS package [58], with constants from Mehrbach et al. [59] as refitted by Dickson and
Millero [60]. Parameters of carbonate SW chemistry in each
treatment are given in Table S1.
EIPA stock solutions (10 mM) were prepared in dimethylsulfoxide and used at a working concentration of 100 lM
(0.1% dimethylsulfoxide) in FSW. Amiloride stock solutions (50 mM) were prepared in FSW and used at a working concentration of 500 lM.
Ammonium chloride stock solution (20 mM) was prepared in FSW or in ASW. Working concentrations (40,
20, 10, 5, 1 and 0 mM) were adjusted to pH 8.2 with
NaOH.
ASW was prepared with 490 mM NaCl, 10 mM CaCl2,
27 mM MgCl2, 29 mM MgSO4, 2 mM NaHCO3 and 10 mM
KCl in distilled water (the pH was adjusted to 8.2 with 1 M
HCl) [61]. Na+-free ASW was prepared with 490 mM choline
chloride, 10 mM CaCl2, 29 mM MgSO4, 27 mM MgCl2,
10 mM KCl, 2 mM choline bicarbonate and 0.5 mM Tris in
distilled water (the pH was adjusted to 8.2 with 1 M HCl) [61].
Analysis of pHi
Symbiont-containing and symbiont-free endoderm cells were
loaded with SNARF-1 by mixing 1 mL of each cell suspension with 2 mL of SNARF-1 AM (Invitrogen, Grand
Island, NY, USA) in FSW (final concentration: 10 lM
SNARF-1 AM, 0.01% pluronic F-127, and 0.1% dimethylsulfoxide). Cells were then incubated in dark conditions for
30 min at 20 °C and washed by 5 min of perfusion with
FSW in the dark to remove residual traces of the dye.
SNARF-1 fluorescence was measured by confocal
microscopy (Leica SP5, Buffalo Grove, IL USA) and calibrated to pHi (NBS scale) with methods published previously [5,19]. Briefly, cells were excited at 543 nm, and
SNARF-1 fluorescence emission was captured in two
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
Regulation of intracellular pH in cnidarians
channels at 585 10 nm and 640 10 nm while transmission was simultaneously monitored. In cells containing
symbionts, the use of 543 nm as the excitation wavelength
minimized chlorophyll autofluorescence, as 543 nm lies outside of the absorption spectrum of chlorophyll a and in a
low region of absorption of the peridinin–chlorophyll–protein complex [62]. pHi image analysis was performed with
LAF-AS software (Leica), with digital regions of interest to
confine fluorescence analysis to the anemone cell cytoplasm,
avoiding dinoflagellate symbionts. The 585/640-nm fluorescence intensity ratio (r) was calculated after subtracting
background fluorescence recorded in a second region of
interest in the surrounding cell medium. r was related to
pHi by the following equation:
pHi ¼ pKa log½r rB =rA r FBðk2Þ =FAðk2Þ where F is fluorescence intensity measured at 640 nm
(k2) and the subscripts A and B represent the values
at the acidic and basic endpoints of the calibration,
respectively. Intracellular calibration of pHi with
SNARF-1 was performed for each experiment in vivo
by ratiometric analysis of SNARF-1 fluorescence in
cells exposed to buffers ranging from pH 6 to pH 8.5
containing the ionophore nigericin [5].
Experimental design
Cells were analyzed in a semi-open perfusion chamber
(PECON, Erbach, Germany) fitted on a temperature-controlled microscope stage (Temperable Insert P, PECON)
maintained at 20 °C in dark conditions. Experiments were
conducted under perfusion (60 mLh1), which kept pH
and TA stable in the SW surrounding the cells during
microscopic observations. Optimization of flow rates and
cell densities to achieve stable pH was performed previously [19], and additional checks of pH and TA were
made in the current investigation by determining pH and
TA in inflowing and outflowing SW from the perfusion
chamber. In all experiments, pHi measurements were
made at 5-min intervals throughout the duration of the
experiment.
Extracellular acidification
Experiments on the impact of SW at pH 7.4 adjusted with
HCl were achieved by first perfusing cells with FSW at the
control pH value of 8.2 for the first 5 min at 60 mLh1.
SW pH in the perfusion chamber was then decreased to 7.4
within 1 min by rapid perfusion with SW at pH 7.4. Perfusion was then resumed at 60 mLh1 at pH 7.4 for the
remainder of the experiment. In experiments with inhibitors, SW pH in the perfusion chamber was then decreased
to 7.4 with FSW containing 100 lM EIPA (0.1% dimethylsulfoxide) or FSW containing 500 lM amiloride.
691
Regulation of intracellular pH in cnidarians
NH4Cl-induced acidification
Two sets of experiments were performed with the ammonium prepulse technique [20–22].
Cells were first perfused with SW at pH 8.2 for an initial
period of 5 min, and then exposed to 20 mM NH4Cl in SW
for 10 min. NH4Cl was then washed out by perfusion with
SW at pH 8.2, which continued until the end of the experiment. In experiments with inhibitors, wash-out SW solutions contained 100 lM EIPA (0.1% dimethylsulfoxide) or
FSW + 500 lM amiloride.
In the second set of experiments, cells were exposed for
the initial 5 min to ASW, and this was followed by 10 min
of exposure to NH4Cl in ASW. NH4Cl was then washed
out by perfusion with ASW or Na+-free ASW at pH 8.2.
Buffering capacity
btotal was determined as previously described, with successive NHþ
4 concentrations [27,50]. Cells were sequentially
exposed to SW containing different concentrations of
NH4Cl (40, 20, 10, 5, 1 and 0 mM) for at least 4 min each
in the presence of 100 lM EIPA. pHi was recorded at each
NH4Cl concentration. btotal was calculated with the following formula provided by Loiselle and Casey (2010) [27]:
btotal ¼
D½NHþ
4 1
DpHi
where ΔpHi represents the change in pHi between each
successive concentration of NH4Cl solution, and Δ[NHþ
4 ]i
represents the intracellular concentration of NHþ
calcu4
lated from pHe, pHi and the external concentration of
NHþ
4 with the following equation [27]:
½NHþ
4 i
ð9:02pHiÞ
½NHþ
4 0 10
¼
ð9:02
pHeÞ
1 þ 10
Analysis of respiration
Samples of 6 mL of FSW containing isolated cells were
divided into two aliquots of 3 mL for analysis of respiration.
The first 3-mL aliquot used for measurements of respiration was transferred to a closed combined plate chamber
(Hydro-Bios, Halifax, NS, Canada). The cell suspension in
the cuvette was agitated with a magnetic stirrer, and was
maintained at 20 0.5 °C with a recirculating water bath
in dark conditions. An oxygen optode sensor system (oxy-4
mini; PreSens, Regensburg, Germany) was used to quantify
oxygen flux after 30 min. The rate of oxygen uptake was
quantified for 5 min. Data were recorded with OXY4V2_11FB
software (PreSens).
The second 3-mL aliquot used for measurement of protein content was first centrifuged (8000 g for 10 min at
4 °C). The pellet was then resuspended in 1 mL of NaOH
692
J. Laurent et al.
(1 M), vortexed, and heated for 10 min at 90 °C for protein
extraction. The BC Assay Protein quantification Kit (Uptima, Montlucßon, France) was used for protein analysis. This
test is a colorimetric method based on interactions between
proteins, copper ions and bicinchoninic acid [63]. The standard curve was established with BSA.
Phylogenetic analysis of NHEs
Sequences for the human and D. melanogaster NHEs were
retrieved from GenBank. These sequences were used as bait
to mine (BLAST) the transcriptome, genome and EST databases of the following marine invertebrates: S. purpuratus
at NCBI, C. gigas at oysterdb.cn, N. vectensis at JGI, and
Ac. digitifera at marinegenomics.oist.jp. The sea urchin
S. purpuratus is a hemichordate (Deuterostomia), the oyster
C. gigas is a mollusc (Protostomia), N. vectensis is a nonsymbiotic sea anemone (Cnidaria), and Ac. digitifera is
symbiotic coral (Cnidaria). All have complete sequenced
genomes and predicted proteomes. Databases were downloaded on a local server and BLAST searched. BLAST-identified
genome and cDNA sequences were manually verified and
corrected when necessary (by the use of genome/cDNA and
genome translation/ortholog alignments). Reverse BLAST
against NCBI_Refseq and SMART domain analysis (smart.embl-heidelberg.de/) validated genuine identification of
the invertebrate NHE homologs. Optimal sequences were
aligned with MAFFT (mafft.cbrc.jp/alignment/server/), with
default parameter (Fig. S2). By use of the conserved portion between positions 467 and 868, a phylogenetic tree
was computed with MRBAYES (mrbayes.sourceforge.net/) on
a local server with no stringent parameter.
Statistical analysis
pHi data were analyzed with SPSS statistical software. Parametric tests were performed when data were normally
distributed. One-way ANOVA and independent-samples
t-tests were performed when there was homogeneity of variances. A Welch ANOVA test was performed when variances were not homogeneous. A Mann–Whitney U-test
(nonparametric) was performed when data were not
normally distributed.
When we compared recovery rates of cells during extracellular acidification experiments and during NH4Cl prepulse experiments, we pooled data for symbiont-containing
and symbiont-free cells, as no significant differences were
found between these groups. An independent-samples t-test
was used to compare recovery rates between the extracellular acidification and NH4Cl prepulse treatments.
Acknowledgements
We thank N. Techer and N. Segonds for their technical
help, and three anonymous referees for their reviews.
FEBS Journal 281 (2014) 683–695 ª 2013 FEBS
J. Laurent et al.
This study was conducted as part of the Centre Scientifique de Monaco Research Program, supported by the
Government of the Principality of Monaco. J. Laurent
was supported by a fellowship from the Centre Scientifique de Monaco. We also thank J. Pouyssegur and J.
Casey for fruitful discussions.
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Supporting information
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Table S1. Carbonate chemistry parameters of different
pH treatments.
Fig. S1. Bayesian phylogenetic tree of NHE homologs
in humans (Hs), Drosophila melanogaster (Dm), the
marine invertebrates Strongylocentrotus purpuratus
(Spu) and Crassostera gigas (OYG), the anemone Nematostella vectensis (Nv), and the symbiotic coral Acropora digitifera (Adi).
Fig. S2. Alignment of the NHEs used for the phylogenetic analysis.
Fig. S3. Respiration rate (mean SEM of six experiments) of anemone endoderm cells subjected to control
treatment (pH 8.2) and acidification treatment
(pH 7.4).
695