Recent advances in understanding trans-epithelial acid-base regulation and excretion mechanisms in cephalopods

Abstract

Cephalopods have evolved complex sensory systems and an active lifestyle to compete with fish for similar resources in the marine environment. Their highly active lifestyle and their extensive protein metabolism has led to substantial acid-base regulatory abilities enabling these organisms to cope with CO₂ induced acid-base disturbances. In convergence to teleost, cephalopods possess an ontogeny-dependent shift in ion-regulatory epithelia with epidermal ionocytes being the major site of embryonic acid-base regulation and ammonia excretion, while gill epithelia take these functions in adults. Although the basic morphology and excretory function of gill epithelia in cephalopods were outlined almost half a century ago, modern immunohistological and molecular techniques are bringing new insights to the mechanistic basis of acid-base regulation and excretion of nitrogenous waste products (e.g. NH₃/NH₄⁺) across ion regulatory epithelia of cephalopods. Using cephalopods as an invertebrate model, recent findings reveal partly conserved mechanisms but also novel aspects of acid-base regulation and nitrogen excretion in these exclusively marine animals. Comparative studies using a range of marine invertebrates will create a novel and exciting research direction addressing the evolution of pH regulatory and excretory systems.

Introduction

Cephalopod biology

Among all invertebrates cephalopods have probably reached the highest degree of complexity, in terms of sensory and locomotive ability. Cephalopods exhibit a high degree of cephalization and a complex neural system with very efficient sensory organs such as lens eyes, chemo-receptors and balance receptors.^1,2 It is believed that these vertebrate-like features arose from the competition with fish since their first occurrence during the so called “Cambrian explosion” around 500 million years ago.^3,4 Although many features of fish and cephalopods have been described as convergent, fundamental anatomical and physiological differences of cephalopods constrain their evolutionary competition with fish. For example most cephalopods, including squid, cuttlefish and octopods use jet propulsion to generate high swimming speeds which is a less efficient swimming mode associated with high energetic costs compared to undulatory swimming movements of fish.^5,6 Although a considerable evolutionary refinement of hemocyanin-oxygen transport has occurred within the cephalopoda this respiratory pigment can only carry about half the oxygen of the cellular hemoglobin of vertebrates.⁷ Thus, in order to optimize the transport efficiency of this pigment, cephalopod hemocyanins usually have a large Bohr-effect increasing their pH sensitivity.⁸ Accordingly cephalopods require a tightly regulated blood pH homeostasis in order to protect gas transport via hemocyanins.^9,10 Additionally, as a trade-off to their less efficient swimming mode and active lifestyle, cephalopods are confronted with strong CO₂ induced temporal acid–base disturbances during jetting and fast swimming. To accommodate such temporal pH fluctuations cephalopods have evolved moderate to strong acid–base regulatory abilities to stabilize blood pH during exercise and hypercapnic exposure.^11-13 Accordingly, well developed acid-base regulatory abilities seem to represent another feature that underlines the convergence of cephalopods and other active marine organisms including fish and crustaceans.

Acid-base physiology in marine vertebrates and invertebrates

Most aquatic organisms stabilize extracellular pH by actively secreting H⁺ and accumulating bicarbonate in body fluids in order to buffer the excess of protons generated through a respiratory or metabolic acidosis.^14-17 Active marine organisms like fish, crustaceans and cephalopods were identified as good regulators in response to environmental hypercapnia and their acid–base regulatory abilities have been well documented.^18,19

For example, in response to 1 kPa CO₂ the marine teleosts Gadus morhua (cod) and Conger conger (eel) initially compensated for an extracellular acidosis by actively accumulating HCO₃⁻ from 10 to 30 mM within 25 h (cod) and from 5 to 22 mM (eel), respectively.^20,21 Studies using decapod crustaceans demonstrated that these invertebrates can also fully compensate hypercapnia induced pH_e disturbances through active bicarbonate accumulation in body fluids.^22-25 For example, in response to 1 kPa water pCO₂ Cancer magister increased its blood [HCO₃⁻] by 12 mM within 24 h to fully compensate pH_e.²⁵ Studies on Carcinus maenas and Callinectes sapidus also indicated comparable high acid–base regulatory abilities.^26-29 Although less active marine invertebrates were generally shown to have lower capabilities to compensate for extracellular acid-base disturbances, some echinoderms including sea urchins (Strongylocentrotus droebachiensis) and brittle stars (Amphiura filiformis) were demonstrated to be able to control extracellular pH to a certain degree.^30,31 The acid-base compensatory mechanism in these species is predominantly achieved through the accumulation of bicarbonate in body fluids, as well as an increased excretion of protons and/or proton equivalents (NH₄⁺).^30,31

Similar to fish and crustaceans, also adult cuttlefish (Sepia officinalis) and squid (Sepioteuthis lessoniana) can compensate for an extracellular acidosis when exposed to elevated environmental pCO₂ by actively increasing blood [HCO₃⁻] levels. The compensation reaction happens within few hours and can lead to a partial compensation in S. officinalis accompanied by an increase in blood [HCO₃⁻] from 3.4 to 10.4 mM¹³ or a full compensation of blood pH as found in S. lessoniana with an increase in blood [HCO₃⁻] from 2.3 to 4.3 mM.¹² Although acid-base regulatory abilities of cephalopods were investigated in great detail,^10,11,13 the responsible organs and mechanistic basis for extracellular pH regulation were just recently identified and characterized.

Identification of ion-regulation and excretory organs in cephalopods: a historical perspective

Gill and gut epithelia are the predominant sites of ion and acid-base regulation in fish and the research on osmoregulation in marine fish dates back to the late 1920s.³² While gut epithelia are mainly important for the secretion of HCO₃⁻ and CaCO₃ precipitation as well as water homeostasis^33,34 specialized cells in gill epithelia, so called mitochondrion rich cells (MRCs) were identified as the main sites of ionic regulation and proton secretion in teleost.³⁵ There was less interest in understanding ion/pH regulatory mechanisms in cephalopods probably due to the fact that cephalopods were generally described as weak osmo-regulators that can only exist in the marine environment.³⁶ Only very few myopsid squids species like Lolliguncula brevis were described to be able to tolerate lower salinities in brackish waters.³⁷ Most studies addressing ion regulation and excretion in cephalopods are from the 70 ties and 80 ties focusing on the exploration of nitrogen excretion pathways in these ammonotelic molluscs.^38-43 Important studies by R. Schipp and H.H. Donaubauer identified the ultrastructural morphology of cephalopod excretory organs, including renal appendages, branchial hearts and gills.^38,39,44 Further studies by Potts and colleagues⁴⁰ identified and characterized the gill as major site of NH₄⁺ excretion in cephalopods. These pioneer studies mainly focused on the physiology of adults leaving a large knowledge gap for early life stages.

During the last decade the question regarding acid-base regulatory abilities and mechanisms in marine species has experienced a considerable revival in the context of environmental change and particularly the acidification of oceans.⁴⁵ Anthropogenic CO₂ emissions are predicted to lead to a rise in surface ocean pCO₂ from 0.04 kPa up to 0.08 – 0.14 kPa within this century.⁴⁶ The increased hydration of CO₂ changes seawater chemistry, causing a drop in ocean pH. While some marine species have been identified as rather sensitive (e.g., less active calcifying species such as corals or echinoderms)^30,47,48 others, mostly active species such as adult fish, crustaceans and cephalopods can tolerate high CO₂ concentrations over long exposure times.^49,50 A major hypothesis that had to be tested was, if the ability to compensate for a hypercapnia - induced acidosis by actively accumulating bicarbonate and eliminating protons from their body fluids is a general, unifying feature of tolerant organisms.⁴⁵ Thus, a first step was dedicated to the identification of acid-base regulatory structures in marine species and the characterization of the underlying ion-regulatory machinery including ion transporters and channels. The advances in molecular biology and available genomes and transcriptomes of the 21^st century have opened new venues to re-address the mechanisms of ion / pH regulation and excretion in non-model species like cephalopods. Thus, the present review aims at summarizing recent advances that have led to a better understanding of acid-base physiology and nitrogen excretion in cephalopods.

Cephalopod Ion-Regulatory Epithelia

Epidermal ionocytes

Cephalopod embryos have an oviparous developmental mode inside an egg capsule which protects the embryo from environmental stressors. On the opposite site this egg capsule constitutes a diffusion barrier for gases such as O₂ and CO₂, with diffusion coefficients of 10–20 % to that of pure seawater, leading to increasing hypoxic and hypercapnic conditions inside the egg along embryogenesis^49,51,52 (Fig. 1A). This prompted us to formulate the hypothesis that already in the embryonic phase, when adult-like ion-regulatory structures are still absent, the developing embryo must have alternative structures to regulate extracellular pH. The cephalopod embryo has an external yolk sac that is well perfused, and pulsatory movements of the yolk drive a circulatory current of blood through the embryonic circulatory system that consists of large blood lacunae mainly in the head region (depicted in Fig. 1A). Immunohistological, ultra-structural, electro-physiological and expression studies confirmed the hypothesis and demonstrated that the yolk epithelium represents an ion regulatory site in cephalopod embryos. Similar to the situation in teleosts, Na⁺/K⁺-ATPase (NKA) rich cells are scattered over the yolk that are equipped with a pH regulatory machinery to mediate extracellular acid-base homeostasis (Fig. 1B).

Figure 1.

Epidermal ionocytes in early life stages of cephalopods developing in a naturally acidified environment. (A) The embryonic micro-environment is characterized by hypoxia, hypercapnia and high ammonia levels caused by a lower diffusion permeability of the protective egg capsule. Abiotic conditions of the perivitelline fluid (PVF) are influenced by environmental changes but also by the developing embryo itself. Depending on the physiological state of the embryo metabolic end-products (e.g., NH₄⁺/NH₃) and protons will be excreted at different rates and will accumulated in the PVF. (B) Epidermal ionocytes of cephalopod early life stages were first discovered using immunohistochemical techniques showing the presence of Na⁺/K⁺- rich cells scattered over yolk and head in squid and cuttlefish embryos adopted and modiefied from. ⁷⁴ Similar to the situation in teleosts the yolk constitutes the largest, well perfused surface area, in the developing embryo, making these epithelia prime sites for gas exchange and ionic regulation before the development of gill epithelia.

Branchial epithelia

The gills of decapod cephalopods like squid and cuttlefish are paired organs with bilateral symmetry, located inside the mantle cavity in a lateral position to the renal sac. A complex folding of the gill consisting of 1^st to 3^rd order lamellae generates a large surface area that is beneficial for gas exchange (Fig. 2). The gills are in direct contact with the water stream inside the mantle cavity, and were suggested to represent important organs mediating the elimination of nitrogenous (NH₄⁺) wastes.^38,39,53,54 The blood is pumped through the gills with the aid of the branchial hearts, contractile blood vessels and muscular movements of the gills.^53,55,56 Inside the gill, the blood enters the primary (1°) afferent vessel bringing deoxygenated blood to the gill which is then sent to the secondary (2°) afferent vessel which is located inside the first order (1°) lamellae and finally distributed to the 3° afferent vessels located in the 2° lamellae. Oxygenation takes place in the blood sinus of the 3° lamellae. After gill passage, oxygenated blood enters the systemic heart and is then distributed back into body tissues via the arterial system.

Figure 2.

General overview of the gill morphology in decapod cephalopods. Diagram of the gill morphology of Sepia officinalis indicating the direction of the blood flow and the arrangement of first to third order lamellae, showing the twice folded second order lamella of the gill. The concave inner epithelium (orange) of the third order gill lamellae belongs to the transport active epithelium, whereas the outer epithelium is exclusively involved in respiratory processes. Drawings modified after.⁵⁵

The complex folding of the cephalopod gill can be separated into 3 different orders of lamellae. The primary lamella is folded in a fanlike pattern to form secondary (2°) lamellae that are aligned at right angles to the axis of the 1° lamella. Each 2° lamella, in turn, is folded in a fanlike pattern to form tertiary (3°) lamellae aligned at right angles to the axis of the 2° lamellae. The 3° gill lamellae consist of 2 epithelial layers lining a blood sinus (Fig. 2). The inner, concave epithelium of the 3° lamellae (Fig. 2 A orange color) is rich in mitochondria and various enzymes (e.g. alkaline phosphatase, acid phosphatase, carbonic anhydrase, Mg²⁺-triphosphatase, β-glucuronidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, succino-dehydrogenase, monoamine oxidase) indicating active transport processes, whereas the thin outer epithelium, is believed to mainly serve respiratory processes.^39,57 These observations suggest that similar to the situation in fish and crustaceans, the cephalopod gill covers multiple functions including gas exchange, acid-base regulation and ammonia excretion.

Development and regulatory pathways for the differentiation of epidermal ionocytes

Before gill epithelia are fully developed, epidermal ionocytes on the skin mediate ion and acid-base balance in teleosts^58-62 (Fig. 1). The process of epidermal development, including the differentiation of ionocytes relies on balanced signaling gradients secreted from non-neural (BMPs) and the neural ectoderm (including the chordin and noggin), and is evolutionarily conserved among vertebrates.^63,64 Under stimulation by BMPs, downstream targets of the transcription factor, ΔNp63, are activated in epidermal stem cells, which thereafter undergo the subsequent process of terminal differentiation.^58,65 So far, in many model teleosts, such as zebrafish and Japanese medaka, these regulatory pathways are controlling the formation of specific epithelial ionocytes during development.^61,66 Furthermore, transcription factors including the forkhead box protein I3 (foxi3) and glial cells missing homolog 2 (gcm2), which transmit the specification and differentiation signals from sending cells (epidermal stem cells) to receiving cells (epidermal ionocytes), were identified as important modulators in teleosts. For example, gain- and loss-of-function experiments demonstrated a specific role for GCM2 in the differentiation process of proton pump-rich (HR).⁶⁷ Recently a partial sequence of ΔNp63 transcription factor was cloned from the cephalopod (Sepia pharaonis) indicating that similar regulatory pathways as those found in vertebrate systems are also present in lophotrochozoans. Preliminary experiments demonstrated the expression of the stem cell marker ΔNp63 in epidermal cells of S. pharaonis (Fig. 3). Thus, future studies addressing the regulatory mechanisms of ionocyte differentiation in cephalopods will represent a fruitful an existing task to explore the evolution of regulatory pathways that control the differentiation of epidermal ionocytes.

Figure 3.

Expression of ΔNp63 in epidermal cells. The stem cell marker ΔNp63 is expressed in epidermal cells of the cuttlefish embryo. Positive signals are found in single epidermal cells scattered over the yolk and skin epithelia. Arrows indicate the location of positively stained epidermal cells.

The pH Regulatory Machinery

The Na⁺/K⁺-ATPase in cephalopod epithelia

The Na⁺/K⁺-ATPase (NKA) is a universal enzyme found in all animal cells, and it generates an electrochemical gradient that can be used by other secondary active transporters.⁶⁸ In the context of osmotic regulation in aquatic organisms the NKA was first demonstrated to be an essential trans-epithelial transport enzyme in fish gills (for a review see).⁶⁹ The role of the NKA in regulating osmotic disturbances in crustaceans was first investigated using the blue crab, Callinectes sapidus acclimated to low salinities.⁷⁰ Further studies identified the gill as the most important site of osmotic regulation and discovered transporters and mechanisms that allowed some crustacean species to actively regulate extracellular osmolarity against steep osmotic gradients.^71,72 Although the existence of the NKA located in branchial epithelia of the cuttlefish Sepia officinalis was revealed using enzymatic assays in the late 70 ties, its role in branchial ion regulation was less well understood.³⁹ Molecular characterization of the cephalopod NKA using full length clones of the squid (Loligo opalescens and L. pealeii) NKA revealed fundamental structural differences between osmo-conformers like cephalopods and vertebrates that are capable of maintaining hypo-osmotic conditions in marine environments.⁷³ The comparatively high Na⁺ concentrations in extracellular fluids of squids (450 mM) compared to lower (100–150 mM) blood Na⁺ concentrations in vertebrates have led to a modification in the charge of amino acids located in the ring at the “external mouth” of the NKA. More positive charges at the mouth of the NKA reduce its sensitivity for external Na⁺ and thus enable the enzyme to achieve optimum function in the marine environment.⁷³

During ontogeny of the cuttlefish S. officinalis the activity of the NKA measured in whole animal homogenates increases exponentially in the early embryonic phase and reaches its maximum activity toward hatch of the paralarva/juvenile animal.⁷⁴ In squid, cuttlefish and octopods high concentrations of NKA were found in different tissues including neurons, renal appendages, branchial heart appendages and gills^38,75,76 (Fig. 4). The latter 3 were believed to mainly serve excretory functions, and the NKA being the major driving force to excrete nitrogenous waste products from body fluids.³⁸ Additionally, recent studies provided compelling evidence that the NKA of cephalopods is also a major player in regulating acid-base homeostasis.^12,49 In cephalopods including squid, cuttlefish and octopus, the NKA is present in high concentrations in gill epithelia located in basolateral membranes providing an electrochemical gradient that is used by a variety of secondary active transporters and ion channels.

Figure 4.

Na⁺/K⁺-ATPase in branchial epithelia of cephalopods. Immunohistological detection of Na⁺/K⁺-ATPase (NKA) in gill tissues of squid (Sepioteuthis lessoniana), cuttlefish (Sepia officinalis) and octopus (Octopus vulgaris). Note the homology of NKA occurrence in the concave inner epithelium of gill lamellae in S. lessoniana and S. officinalis. Despite a different gill morphology in O. vulgaris, positive NKA immunoreactivity is also predominantly found in concave (semi-tubular) parts of the gill epithelium.

The V-type H⁺-ATPase in cephalopod ion regulatory epithelia

The vacuolar type H⁺-ATPase (VHA) is a highly conserved enzyme that covers a remarkable diversity of functions in eukaryotes (for a review see).⁷⁷ Besides its role in acidifying intracellular organelles, it is well known to transport protons across the plasma membrane of specialized cells to regulate extra - cellular pH homeostasis.^77-80 Due to its structure with the catalytic (V1 complex) site in the cytosol the VHA is restricted to pump H⁺ out of the cytoplasm, and thus cannot function as a direct pathway to load a cell with protons. The VHA has been demonstrated to be highly expressed in most vertebrate and invertebrate ion regulatory epithelia including kidneys,⁷⁸ skin and gills.^{74,75,79,81,82} In most epithelia contributing to extracellular pH regulation the VHA is expressed in apical membranes contributing to the secretion of protons from the organism.^78,81 However, a basolateral orientation of the VHA has been associated with base secretory processes through cooperating with apical pendrin (SLC26A4) to secrete base in type B intercalated cells of the mammalian kidney^78,83 or in branchial epithelia of some elasmobranchs.^84,85 Also in dipteran insect larvae, a basal V-ATPase that is coupled to apical anion exchange is responsible for a net import of HCO₃⁻ into the luminal space to drive luminal alkalinization.^86,87 Also in euryhaline killifish, Japanese medaka (Oryzias latipes), a basolateral VHA is found in ionocytes co-expressing the Na⁺-Cl⁻ cotransporter (NCC).⁶⁰ And NCC type ionocytes in tilapia express apical NCC and basolateral Na⁺/HCO₃⁻ co-transporter (NBCe1), which was postulated to mediate Na⁺ uptake and acid–base regulation.⁸⁸ Therefore the basolateral VHA expressed in NCC type ionocytes may be involved in NaCl uptake as well as acid–base regulation. Immunohistochemical analyses using antibodies specifically designed for squid (S. lessoniana) VHA could demonstrate that also in cephalopods the VHA is restricted to basolateral membranes in gill epithelia as well as epidermal ionocytes of embryonic stages.^12,89 Interestingly, positive VHA immunoractivity was additionally found in pilaster (or pillar) cells spanning through the blood sinus between the inner and the outer epithelium of the cephalopod gill. Expression studies demonstrated an up-regulation of the VHA (summarized in Table 1) in response to hypercapnia (acidification) indicating that this enzyme must be involved in regulating pH homeostasis during hypercapnic exposure in cephalopods. It was suggested that the basolateral orientation of the VHA in ionocytes and gill epithelia, as well as its expression in pillar cells may contribute to a local acidification of the blood to optimize gas exchange and/or facilitate the formation of the ammonium ion (NH₄⁺) that can be used as an alternative substrate by the NKA.¹² To date little information exists regarding the function of branchial pillar cells in aquatic organisms. Pillar cells characterized by high concentrations of extracellular membrane bound carbonic anhydrase IV were found in the dogfish Squalus acanthias, and were associated with gas exchange processes by facilitating the formation of CO₂.⁹⁰ Accordingly, it can be hypothesized that similar to the situation in dogfish, VHA localized in pillar cells of squid gills may also support CO₂ excretion during hypercapnic conditions by providing protons for the formation of CO₂.

Table 1.

Gene expression changes in different ontogenetic stages of the squid Sepioteuthis lessoniana during short-term and prolonged acclimation to hypercapnic conditions (0.4 kPa pCO₂)

Future studies determining epithelial fluxes of acid-base equivalents in combination with specific inhibitors are needed in order to better understand the role of the VHA in cephalopod ion-regulatory epithelia.

The cephalopod acid-base regulatory machinery

In convergence to fish and crustaceans, cephalopods evolved branchial ion regulatory epithelia, which are equipped with ion transporters beneficial for coping with acid–base disturbances. Cloning strategies using degenerate primers designed against highly conserved regions of the NKA, VHA, Na⁺/H⁺-exchangers (NHEs), carbonic anhydrase (CA) and Na⁺/HCO₃⁻ co-transporters (NBC_e) resulted in partial DNA sequences encoding these enzymes in squid and cuttlefish.^12,49 In situ hybridization and immunohistochemical methods with antibodies specifically designed against acid-base transporters of squid, revealed the sub-cellular localization of these enzymes in cephalopod ion-regulatory epithelia.^12,49 For example Na⁺/H⁺ exchanger 3 (NHE3) which is an essential player for proton secretion in vertebrates^78,91-93 is also expressed in apical membranes of epidermal ionocytes and gill epithelia of squid (S. lessoniana) and cuttlefish (S. oficinalis) (Fig. 5A).^12,74,89 Proton selective electrodes in combination with 5-ethylisopropyl amiloride (EIPA) a specific inhibitor for NHE proteins could demonstrate that at least 60 % of H⁺ secretion across the surface of epidermal ionocytes of embryonic squids is mediated through Na⁺/H⁺ antiport.⁷⁴ The involvement of NHEs instead of proton pumps like the VHA to mediate apical proton secretion in marine species is thermodynamically favored by the strong Na⁺ gradient between cytosol (30 mM) and seawater (470 mM).^94,95 It was hypothesized that NHE based proton secretion may represent a common pathway in marine organisms which however needs further confirmation by testing more marine species including crustaceans, echinoderms, annelids, and mollusks. Besides the secretion of protons, cephalopods including squid and cuttlefish were demonstrated to actively accumulate HCO₃⁻ to stabilize blood pH during an acidosis.^12,13 In vertebrate systems the basolateral import of HCO₃⁻ is achieved by various transporters of the SLC4 family, including anion exchangers, and Na⁺/HCO₃⁻ co-transporters.^96,97 Two potential candidates for the transport of HCO₃⁻ ions including an electrogenic Na⁺/HCO₃⁻ co-transporter (NBC_e) and a Na⁺-dependent Cl⁻/HCO₃⁻ exchanger (NDCBE) were cloned from the squid giant axon.^98,99 While NDCBE had comparatively low transcript abundance in branchial epithelia but higher RNA levels in neurons, the electrogenic NBC was highly expressed in gill epithelia and expression of this gene was stimulated upon hypercapnic exposure.^12,49,99 Another important enzyme group in the context of acid-base balance are carbonic anhydrases that catalyze the formation of HCO₃⁻ from water and CO₂.¹⁰⁰ Besides their function in vertebrate systems^69,101,102 carbonic anhydrases were also demonstrated to play an important role in the acid-base regulatory mechanisms of aquatic invertebrates (e.g., crustaceans).^103,104 To date, studies addressing the role of carbonic anhydrases in mediating acid-base homeostasis in cephalopods are still underrepresented. Earlier studies by Schipp and colleagues³⁹ located a cytosolic carbonic anhydrase in the transporting epithelium of the cuttlefish gill using histochemical methods. Using degenerate primers a partial sequence of a cytosolic carbonic anhydrase isoform was cloned from cuttlefish and squid gills.⁴⁹ This gene also responded with increased transcript levels in gill epithelia of squid (S. lessoniana) upon acute (<24 h) exposure to moderate acidification levels of pH 7.7.¹² According to this information a first working model for the cephalopod branchial acid-base regulatory machinery during hypercapnic acclimation was suggested (Fig. 5B). In response to a hypercapnia induced acidosis the drop is blood pH is accompanied by an increase in pCO₂ in order to maintain a sufficient outward directed CO₂ gradient. CO₂ can easily diffuse across biological membranes, and its subsequent hydration in the cytosol catalyzed by a cytosolic CA, leads to the formation of HCO₃⁻ ions and protons. The proton generated in this process is exported across the apical membrane via NHE3, while the bicarbonate is transported across the basolateral membrane, where it is accumulated in the blood to buffer the excess of protons. The secretion of protons via apical Na⁺/H⁺-exchanger is driven by the high environmental [Na⁺] while basolateral HCO₃⁻ absorption has been suggested to be achieved by an electrogenic Na⁺/HCO₃⁻ co-transporter driven by the intracellular formation of HCO₃⁻ catalized by CA and a negative membrane potential created by the Na⁺/K⁺-ATPase.

Figure 5.

The branchial acid-base regulatory machinery. Immunohistochemical localization of NHE3 in apical membranes of the cephalopod (Sepioteuthis lessoniana) gill facing the semi-tubular space of the 3^rd order gill lamellae (A). Hypothetical model for acid-base relevant ion transport in the cephalopod gill, including apical proton secretion and basolateral HCO₃⁻ import (B).

Coupling of acid-base regulation and NH₄⁺ excretion

Recent advances in understanding the mechanistic basis for extracellular pH regulation in cephalopods identified gill epithelia as probably the most important regulatory site in adult cephalopods. Additionally, gill epithelia of cephalopods including cuttlefish and octopods were suggested to represent the major site for the excretion of nitrogenous waste products.^39,40 An efficient NH₄⁺ regulatory machinery is probably very important for cephalopods as these animals are highly ammonotelic, exclusively fueling their active lifestyle through protein metabolism.^54,105 In vertebrate systems Rh glycoproteins including Rhbg and Rhcg were identified as central players facilitating the export of ammonia (NH₃) in concert with proton secretion mechanisms allowing a NH₄⁺ trapping mechanism in the acidified boundary layer of excretory cells.^106-109 Just lately Rh proteins were also found and characterized in a range of invertebrates including crustaceans and insects.^110-112 Also in cephalopods one Rh-protein (RhP) has been identified, which has the highest degree of identity to RhPs cloned from the decapod crustaceans Carcinus maenas (50%) and Metacarcinus magister (56 %). Phylogenetic analyses based on amino acid identities demonstrated that the invertebrate specific RhP1-clade clearly separates from vertebrate Rh proteins including RhBG, RhCG, RhAG clades as well as RhP2 and Rh30 clades.⁸⁹

In various cephalopod species the largest fraction of ammonia is produced through amino acid metabolism. Some midwater cephalopods (e.g. cranciidae) store NH₄⁺ in the mM range in coelomic chambers (e.g., cranchiidae) while other ammoniacal species store it in specialized vacuoles within muscle tissues to support neutral buoyancy.¹¹³ Although exchange of Na⁺ against NH₄⁺ can serve to improve neutral buoyancy, most shallow water cephalopods actively secrete ammonia via branchial epithelia.^40,54 Here it was hypothesized that ammonia is excreted as ammonia (NH₃) accompanied with an excretion of protons to form the ammonium ion (NH₄⁺).⁴⁰ The apical localization of RhP in the cephalopod gill epithelia supports earlier studies suggesting the cephalopod gill to be the major site of NH₄⁺ excretion. Moreover, co-localization of NHE3 and RhP strongly suggests an acid-trapping mechanism of NH₄⁺ similar to that demonstrated for diverse vertebrate excretory organs including skin, gill and kidneys.^107,114,115 For example in acid-secreting intercalated cells of the mammalian kidney acid-secretion is coupled to the export of ammonium ions that are trapped in the acidified urine.¹⁰⁶ Here, the basolateral localization of Rhbg and Rhcg is believed to facilitate the entry of the de-protonized ammonia (NH₃) from the blood into the cell where the majority of NH₃ is protonized to the ammonium ion at a physiological pH of approximately 7.2. Apical excretion and subsequent trapping of NH₄⁺ in the urinary space of the collecting duct is achieved by apical Rhcg and proton pumps like the VHA and the H⁺/K⁺-ATPase.¹¹⁶ Interestingly, the gills of coleoid cephalopods show striking morphological and functional similarities to the collecting duct of the mammalian kidney. The 3^rd order lamellae of the cephalopod gill form a semi-tubular structure creating a luminal space into which NH₄⁺ is secreted by the interplay of RhP and NHE3 (Fig. 6 A, B). At the basolateral membrane the entry of the ammonium ion (NH₄⁺) is achieved by the NKA that can also accept NH₄⁺ as a substrate. Here it remains to be investigated if the microenvironment within the semi-tubular space of the 3^rd order lamellae is also acidified, similar to the mammalian collecting duct. Thus, further studies on pH gradients within the cephalopod gill will help, better understanding the structural and functional convergence between vertebrate and invertebrate excretory systems.

Figure 6.

Identification of NH₄⁺ transport pathways in cephalopod branchial epithelia. Immunohistochemical analyses demonstrate the presence of RhP in apical membranes of the inner epithelium of the third order gill lamellae (A). Hypothetical model for the transport of NH₄⁺ across the branchial epithelium (B). At the basolateral membrane NH₄⁺ is imported into the cell by the NKA. At the apical membrane NH₄⁺ is deprotonated and NH₃ and H⁺ are separately transported into the semi-tubular space where a trapping of NH₄⁺ is proposed.

Role of Ion Regulatory Epithelia During Acclimation to Environmental Hypercapnia

Elevated environmental CO₂ concentrations (hypercapnia) are a stressor for marine species that has lately received considerable attention in the context of ocean acidification.^45,48,117 Environmental hypercapnia that alters positive diffusion gradients of CO₂ from the animal to the seawater will affect the acid-base physiology of all water breathing animals as intra- and extra-cellular CO₂ concentrations will increase as well to maintain a sufficient diffusion gradient to excrete metabolic CO₂.⁴⁵ Among a range of marine species, some have been identified as more sensitive (e.g. less active calcifying species such as corals or echinoderms) whereas others (many active species such as adult fish and cephalopods) can tolerate relatively high CO₂ concentrations over long exposure times. It was hypothesized that the degree of sensitivity is directly linked to a species ability to compensate for acid-base disturbances by actively accumulating bicarbonate and eliminating protons from their body fluids.

Initially, different hypotheses regarding the tolerance of cephalopods toward hypercapnia have been proposed based on their physiological features. On one hand, high Bohr coefficients of the cephalopod hemocyanins were proposed to be a critical physiological characteristic that would make cephalopods particularly sensitive to acid-base disturbances.¹¹⁷ On the other hand substantial acid-base regulatory abilities as found in most cephalopod species represents a common feature that was suggested to make ectothermic marine animals robust to seawater acidification.⁴⁵ However, metabolic data from the cuttlefish S. officinalis (acute exposure to 0.6 kPa pCO₂) and the squids Dosidicus gigas (acute exposure to 0.1 kPa pCO₂) and Sepiteuthis lessoniana (acute; 20 h medium term; 168 h 0.16 and 0.41 kPa pCO₂) indicated that these animals respond very differently toward environmental hypercapnia.^12,118,119 Although these studies partly used unrealistically high pCO₂ levels, in the context of environmental hypercania (usually >0.1 kPa), they could indicate a higher sensitivity in active pelagic squids compared to cuttlefish that have a benthic lifestyle. It has been suggested that different lifestyles and energetic limitations could represent a major feature that limits the ability to mobilize energy resources to fuel acid-base compensatory processes in different cephalopod species.¹² The increased energetic costs during acclimation to acidified (hypercapnic) conditions are reflected in the up-regulation of important ion transporters and pumps, expressed in branchial epithelia of cephalopods (see Table 1). Although, modulations on mRNA do not necessarily show a strong correlation to protein levels, yet changes in transcript abundance indicate an altered transcriptional turnover of the respective transporter triggered through changes in seawater acidity and/or pH. However, more studies using cephalopod species with different lifestyles and energetic requirements are needed in order to provide more conclusive answers regarding characteristic features that determine the degree of sensitivity toward acid-base disturbances.

Proton secretion and HCO₃⁻ buffering

During acclimation to hypercapnic conditions cephalopods including squid and cuttlefish were shown to actively increase HCO₃⁻ levels in their blood in order to buffer the excess of protons generated through the increased hydration of CO₂.^12,13 The buffering of protons happens in concert with the direct secretion of protons or proton equivalents from the animal via specialized cells located in regulatory epithelia (see previous section). Interestingly, the ratio between buffering and proton secretion can vary greatly between cephalopod species. For example under control conditions venous HCO₃⁻ and pHe levels of S. lessoniana (2.5 mM; pH 7.35) were found to be in the range as described for other cephalopod species including the squid Illex illecebrosus (2.2 mM; pH 7.46) and the cuttlefish Sepia officinalis (3.4 mM; 7.70).^11–13 Differences in extracellular pH under control conditions may be attributed to the higher activity level of pelagic squids (S. lessoniana and I. illecebrosus) as well as slightly lower (1 mM) blood [HCO₃⁻] compared to the cuttlefish S. officinalis. Furthermore, when squid (S. lessoniana) and cuttlefish (S. officinalis) were exposed to similar acidification levels of pH 7.1 (0.6 kPa pCO₂) to 7.3 (0.4 kPa pCO₂) blood HCO₃⁻ levels in S. officinalis increased by approximately 7.5 mM within 48 h¹³ whereas in S. lessoniana blood [HCO₃⁻] were only increased by 2 mM within the same acclimation period.¹² These findings indicate that different pH buffering/regulatory mechanisms, including non-bicarbonate buffering and H⁺ extrusion mechanisms exist among cephalopods. Together with the fact that non-bicarbonate buffer values of squid blood (4.7 to 5.8 mmol l⁻¹ pH unit⁻¹) are generally lower than those determined for cuttlefish (10 mmol l⁻¹ pH unit⁻¹) it can be suggested that extracellular acid-base regulatory mechanisms in squids rely on active proton extrusion mechanisms to a greater extend than on buffering strategies. In this context, the coupling of NH₄⁺ excretion and proton secretion could represent a fundamental pathway to directly excrete protons from extracellular fluids in marine invertebrates.^{12,29,112,120,121} This hypothesis is supported by the fact that a range of marine species including molluscs,¹²¹ echinoderms^30,31 and crustaceans²⁹ increase NH₄⁺ excretion rates in response to acidified conditions. Recently established perfusion techniques using isolated gills of octopus represent a technical breakthrough to study pH regulation (e.g., proton transport rates v.s. HCO₃⁻ transport rates) in cephalopod epithelia. These studies will provide an important basis for future research and will help to improve our understanding regarding different acid-base regulatory strategies in cephalopods.

Temporal effects on the expression of acid-base transporters upon exposure to environmental hypercapnia

In squid and cuttlefish the compensation of extracellular pH during acclimation to environmental hypercapnia is accompanied by dynamic changes in expression levels of acid-base transporters in ion regulatory epithelia. High-resolution time series experiments indicated that the expression of genes coding for acid-base relevant transporters is timely regulated in cephalopods. Among both, adults and embryonic stages, a hypercapnia-induced upregulation of acid-base transporters in ion regulatory epithelia was mainly observed in the acute (<48 h) acclimation phase (Table 1). During prolonged exposure to hypercapnic (acidified) conditions no significant changes were evident when compared to control animals. The increased expression of acid-base transporters during the acute acclimation phase indicates a higher energetic demand for protein synthesis and to fuel the increased number secondary active transporters. This energetically expensive acute acclimation reaction corresponds to a rapid increase in blood HCO₃⁻ levels and a stabilization of extracellular pH close to control levels. Similar observation of a bi-phasic acclimation reaction of acid-base transporters to environmental hypercapnia were observed in teleosts, including Atlantic cod (Gadus morhua)⁵⁰ and eelpout (Zoarces viviparus).¹²² However during prolonged acclimation to hypercapnic conditions, expression levels of acid-base relevant transporters returned back to control levels potentially shifting to an energetically favorable reorganization of physiological features to cope with moderate hypercapnic conditions over longer exposure times. Furthermore, this reorganization of physiological features, including a metabolic shift upon chronic exposure to hypercapnic conditions is essential in order to reduce extra-free radical stress and keep cellular metabolic and redox state in balance.^123-125

Synopsis and Future Perspectives

The recent insights into the ion-regulation physiology of cephalopod molluscs has demonstrated that despite their relatively low ability for osmotic regulation, these animals have evolved sophisticated vertebrate-like acid-base regulatory mechanisms to control extracellular pH homeostasis. Along ontogeny there is a shift from embryonic regulatory epithelia (e.g. yolk epithelium and skin) to adult-type regulatory epithelia (e.g., gills) that enable these organisms to control pH homeostasis during their oviparous embryonic development and after hatch in the marine environment. This ontogenetic shift and sites of ion-regulation once again underline similarities between teleosts and cephalopods indicating a convergent evolutionary trait between these 2 animal groups. Furthermore, the development of cellular, biochemical and molecular techniques helped us to shed light on the epidermal pH regulatory machinery of cephalopods. The identification and characterization of key acid-base transporters demonstrated that the molluscan pH regulatory machinery shows many evolutionary conserved features to those found in vertebrate and mammalian systems. Nonetheless differences do exists, largely due to an exclusively marine and highly ammonotelic lifestyle of cephalopods.

Recently developed perfusion techniques on isolated gill tissues demonstrated that the cephalopod gill is suitable for in vivo examinations of ion-fluxes across the branchial epithelium. These techniques allow an in depth examination of transport pathways using pharmacological approaches, and will broaden our understanding regarding NH₄⁺ -based acid-base regulatory pathways in cephalopods.

Finally, the tightly regulated extracellular pH homeostasis via specialized epithelia requires control and coordination by neural, endocrine and paracrine signaling pathways that are virtually unexplored for cephalopods. However, first investigations suggest that also in cephalopods neuronal control systems using neurophysin peptides (e.g. octopressin and sepiatocin) exist that are involved in the mediation of pH homeostasis through activating the differentiation and proliferation of ionocytes. Thus, hormonal control of acid-base regulation and NH₄⁺ excretion in cephalopods will represent a fruitful future research area. This information together with our recent knowledge will spark light on the evolution of pH regulatory systems in non-model species and will also contribute toward a better understanding of pH regulation in vertebrate and mammalian systems.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.