Regulation of intracellular pH in cnidarians - Wiley Online Library

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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.

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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 684

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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

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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.

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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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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).

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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

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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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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).

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A

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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

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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 688

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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

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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

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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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[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

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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)

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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

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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.

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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

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(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

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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 Additional supporting information may be found in the online version of this article at the publisher’s web site: 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).

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