New insights into the many functions of carbonic anhydrase in fish gills

Respiratory Physiology & Neurobiology 184 (2012) 223–230

Contents lists available at SciVerse ScienceDirect

Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol

Review

New insights into the many functions of carbonic anhydrase in fish gills夽 Kathleen M. Gilmour ∗ Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON, K1N 6N5, Canada

a r t i c l e

i n f o

Article history: Accepted 1 June 2012 Keywords: Acid–base regulation Ionic regulation Chemoreception CO2 excretion

a b s t r a c t Carbonic anhydrase (CA) is a zinc metalloenzyme that catalyzes the reversible reactions of carbon dioxide and water: CO2 + H2 O ↔ H+ + HCO3 − . It has long been recognized that CA is abundant in the fish gill, with attention focused on the role of CA in catalyzing the hydration of CO2 to provide H+ and HCO3 − for the branchial ion transport processes that underlie systemic ionic and acid–base regulation. Recent work has explored the diversity of CA isoforms in the fish gill. By linking these isoforms to different cell types in the gill, and by exploiting the diversity of fish species available for study, this work is increasing our understanding of the many roles that CA plays in the fish gill. In particular, recent work has revealed that fish utilize more than one model of CO2 excretion, that to understand the role of CA and the gill in ionic regulation and acid–base balance means characterizing the transporter and CA complement of individual cell types, and that CA plays roles in branchial sensory mechanisms. The goal of this brief review is to summarize these new developments, while at the same time highlighting key areas in which further research is needed. © 2012 Elsevier B.V. All rights reserved.

1. Introduction It has long been recognized that carbonic anhydrase (CA), the zinc metalloenzyme that catalyzes the reversible reactions of carbon dioxide and water (CO2 + H2 O ↔ HCO3 − + H+ ), is abundant within the fish gill (Sobotka and Kann, 1941; see review by Maren, 1967). Early work focused on the possible contributions of branchial CA to CO2 excretion versus ion transport (e.g. Haswell et al., 1980), two of several physiological processes in which the fish gill plays a key role (Evans et al., 2005). Comparison of the effects of acetazolamide, which inhibits both red blood cell (RBC) and branchial CA, with those of a selectively permeable inhibitor (CL 11,366) that strongly inhibited RBC CA with only a negligible effect on branchial CA, suggested that red cell rather than gill CA was responsible for the dehydration of plasma HCO3 − to CO2 for diffusion across the gill (Maren, 1967). This conclusion was strengthened by later studies that used blood-perfused preparations to document a direct correlation between hematocrit and CO2 excretion, with CO2 excretion being eliminated by plasma or saline perfusion (Perry et al., 1982; see review by Perry, 1986). By contrast, acetazolamide administration was found to inhibit both sodium and chloride uptake across the freshwater fish gill (Maetz

夽 This paper is part of a special issue entitled “New Insights into Structure/Function Relationships in Fish Gills”, guest-edited by William K. Milsom and Steven F. Perry. ∗ Tel.: +1 613 562 5800x6004; fax: +1 613 562 5486. E-mail address: [email protected] 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2012.06.001

and Garcia-Romeu, 1964), suggesting that CA-catalyzed hydration of CO2 within the branchial epithelium provides H+ and HCO3 − that are used as counter-ions for Na+ and Cl− uptake, respectively (Maetz, 1971). Subsequent studies in vivo, in situ and using perfused gill preparations confirmed that branchial CA inhibition resulted in reduced Na+ or Cl− influx (Kerstetter and Kirschner, 1972; Payan et al., 1975; Boisen et al., 2003; Chang and Hwang, 2004). Additionally, a direct relationship between Cl− uptake and saline CO2 tension was demonstrated using a saline-perfused head preparation, underscoring the role of CO2 hydration as the source of H+ and HCO3 − (Perry et al., 1984). The hydration of CO2 that is moving across the gill to provide H+ and HCO3 − for branchial NaCl uptake functionally couples CO2 excretion and ionic regulation, with both of these processes being linked to acid–base regulation. Acid–base regulation in fish relies primarily on modulation of Cl− /HCO3 − and Na+ /H+ exchanges at the gill so as to adjust plasma HCO3 − concentration (Claiborne et al., 2002; Perry et al., 2003; Evans et al., 2005). Clearly CA should contribute to acid–base regulation, but surprisingly few studies have investigated directly the role of branchial CA in acid–base regulation. In dogfish, Squalus acanthias, selective inhibition of branchial (and not RBC) CA activity using benzolamide reduced the rate of excretion of HCO3 − to the water during alkalosis induced by NaHCO3 infusion (Swenson and Maren, 1987). In rainbow trout, Oncorhynchus mykiss, branchial net acid excretion was significantly reduced by acetazolamide treatment, and the effect of acetazolamide treatment was stronger in trout exposed to environmental hypercapnia to cause respiratory acidosis (Georgalis et al., 2006).

224

K.M. Gilmour / Respiratory Physiology & Neurobiology 184 (2012) 223–230

In addition, changes in branchial CA mRNA abundance and/or protein levels have been reported in response to acid–base challenges (Hirata et al., 2003; Georgalis et al., 2006). In recent years, the availability of information on different CA isoforms, the molecular characterization of different cell types in the branchial epithelium, and the development of new experimental models have elaborated upon the roles played by CA in CO2 excretion, ionic regulation and acid–base balance. It has become clear that substantial differences exist among species, that “branchial CA” may in fact constitute more than one CA isoform, and that the expression of CA isoforms within the gill epithelium may be cell-type specific. Keeping in mind the wealth of comprehensive reviews available on the roles of CA in CO2 excretion, ionic regulation, and acid–base balance (recent examples include Henry and Swenson, 2000; Perry and Gilmour, 2006; Esbaugh and Tufts, 2006; Tresguerres et al., 2006a; Hwang and Lee, 2007; Gilmour and Perry, 2009, 2010), the goal of the present review is to summarize the new developments surrounding branchial CA, while at the same time highlighting key areas in which further research is needed.

2. New insights into the role of branchial CA in CO2 excretion As outlined above, the classic model of CO2 excretion across the fish gill posits dehydration of plasma HCO3 − to molecular CO2 within the RBC, catalyzed by RBC cytosolic CA, with subsequent diffusion of molecular CO2 across the branchial epithelium. Bicarbonate ions carried in the plasma access the erythrocyte interior via band 3 anion exchanger in the RBC membrane, and this process is viewed as being the rate-limiting step in CO2 excretion. Branchial CA is not involved because it is intracellular and inaccessible to HCO3 − in the plasma. This model is well supported experimentally for teleost fish (for review see Henry and Heming, 1998; Tufts and Perry, 1998; Henry and Swenson, 2000; Perry and Gilmour, 2002), which appear to lack plasma-accessible branchial CA activity (see Gilmour and Perry, 2009 for a review of this evidence). In two elasmobranch fish (spiny dogfish, S. acanthias, and longnose skate, Raja rhina) and the Pacific hagfish Eptatretus stoutii, however, a range of evidence suggests the presence of membranebound branchial CA that is accessible to plasma HCO3 − , and in the specific case of the spiny dogfish, contributes in a substantial fashion to CO2 excretion (for review see Gilmour and Perry, 2010). The gill in these fish contains significant levels of membrane-associated CA activity that can be released from its membrane linkage by the enzyme phosphatidylinositol phospholipase C, indicating the presence of a glycosylphosphatidylinositol (GPI) anchor (Gilmour et al., 1997, 2002; Esbaugh et al., 2009). This CA activity is also sodium dodecylsulfate (SDS) resistant, consistent with the presence in the mature protein of disulfide linkages (Gilmour et al., 1997, 2002; Esbaugh et al., 2009). These traits are characteristic of the mammalian membrane-bound CA isoforms IV and XV (Hilvo et al., 2005), and type IV-like CAs were cloned from the gill of dogfish (Gilmour et al., 2007) and hagfish (Esbaugh et al., 2009). Dogfish CA IV was localized by in situ hybridization and immunohistochemisty to the plasma membranes of gill pillar cells, a location in which it would be accessible to plasma HCO3 − reactions (Gilmour et al., 2007). Low doses of benzolamide were used in vivo in dogfish to assess the contribution of extracellular CA activity to CO2 excretion (Gilmour et al., 2001). Benzolamide permeates cell membranes slowly, so low doses of the drug for short periods of time provide a means of selectively inhibiting extracellular CA activity without affecting branchial or RBC cytosolic CA isoforms (see Supuran and Scozzafava, 2004; Gilmour and Perry, 2010). Selective inhibition of extracellular CA activity in vivo reduced HCO3 − clearance from the blood during passage through the gill, raising the partial

Fig. 1. A schematic representation of CO2 excretion and base excretion at the dogfish (Squalus acanthias) gill. Cytosolic CA is found within the gill epithelium (Tresguerres et al., 2007), where it contributes to base excretion by catalyzing the hydration of CO2 to provide HCO3 − that is exported across the apical membrane via a pendrintype anion exchanger (Piermarini et al., 2002), and H+ that is exported across the basolateral membrane via H+ -ATPase. By facilitating the conversion of CO2 to HCO3 − within the cell, this CA also functions as a key element of the sensory mechanism that detects metabolic alkalosis and activates sAC to promote H+ -ATPase translocation to the basolateral membrane (Tresguerres et al., 2007, 2010). Cytosolic CA within the RBC contributes to CO2 excretion, as does pillar cell CA IV (reviewed by Gilmour and Perry, 2010). Electroneutral exchangers are drawn as open circles while ATPases are drawn as filled circles. The detection of metabolic alkalosis and activation of sAC and H+ -ATPase translocation are indicated by the unfilled arrows. P, pendrin-like anion exchanger; CAc, cytosolic carbonic anhydrase; CA IV, dogfish carbonic anhydrase IV; sAC, soluble adenylyl cyclase; AE, band 3 anion exchanger; Hb, hemoglobin; RBC, red blood cell.

pressure of CO2 in the arterial blood (PaCO2 ) and causing acidosis (Gilmour et al., 2001). These findings support the involvement of branchial extracellular CA activity in CO2 excretion in dogfish, as does the observation that neither RBC removal (Gilmour and Perry, 2004) nor use of anion exchange inhibitors to block access of plasma HCO3 − to RBC cytosolic CA, impairs CO2 excretion (Gilmour et al., 2001). Reducing hematocrit in vivo to 5% or treating fish with 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) had no impact on PaCO2 , arterial pH and/or HCO3 − clearance at the gill in dogfish unless extracellular CA was also inhibited (Gilmour et al., 2001; Gilmour and Perry, 2004). Collectively, these data suggest that branchial CA IV plays a significant role in CO2 excretion in dogfish. Why is extracellular HCO3 − dehydration an important contributor to CO2 excretion in dogfish (Fig. 1) but not in teleost fish, which lack branchial plasma-accessible CA altogether, or mammals, where pulmonary capillary endothelial CA IV is present but makes a negligible contribution to CO2 excretion (for review see Henry and Swenson, 2000; Swenson, 2000)? Ultimately, this question speaks to the evolution of different pathways of CO2 excretion in different vertebrate groups – why does the diversity of pathways exist, and what are the benefits of the various strategies? Reconstructing the evolution of physiological pathways is not straightforward (see, for example, Berenbrink et al., 2005), and in the case of CA and CO2 excretion, the paucity of data for entire vertebrate groups (e.g. sarcopterygian fish, amphibians, reptiles, birds) is a further complication. However, although the ultimate causes of differences in CO2 excretion pathways remain to be determined, we can begin to dissect the proximate mechanisms through which

K.M. Gilmour / Respiratory Physiology & Neurobiology 184 (2012) 223–230

differences in CO2 excretion pathways arise (see also Gilmour and Perry, 2010). In teleost fish and mammals, the RBC is a far more favorable environment than plasma for HCO3 − dehydration owing to higher CA activity and higher buffering capacity; higher buffering capacity both ensures the proton supply for HCO3 − dehydration and increases the catalytic efficiency of CA (Henry and Swenson, 2000). The CA activity and buffering capacity in mammalian RBCs are, for example, 100-fold and 10-fold higher, respectively, than in the plasma, driving the majority of HCO3 − dehydration through the RBC despite the presence of pulmonary capillary endothelial CA IV (Henry and Swenson, 2000; Swenson, 2000). By contrast, dogfish RBCs exhibit HCO3 − dehydration rates that are lower than those of at least some teleost fish (Perry et al., 1996). A slower CA isoform (low kcat or turnover number) and relatively low CA concentration (Maren et al., 1980; Swenson and Maren, 1987) combine to yield low RBC CA activity (Henry et al., 1997). In addition, the absence of a Haldane effect means that RBC HCO3 − dehydration does not benefit from the release of oxylabile protons during hemoglobin oxygenation (Wood et al., 1994; Perry et al., 1996). These factors lessen the advantage of the RBC as the site of HCO3 − dehydration, while at the same time, the unusually high buffering capacity of dogfish plasma relative to whole blood (Gilmour et al., 2002) overcomes the standard limitation of plasma as a site of HCO3 − dehydration by ensuring the necessary supply of protons. The net effect is to increase the importance of plasma HCO3 − dehydration relative to that in the RBC. Presumably, a similar situation exists in other elasmobranch fish, although data to support this assertion are sparse. The elasmobranch fish that have been examined lack a Haldane effect (Butler and Metcalfe, 1988), and exhibit high plasma buffering capacity (Gilmour et al., 2002), but more information is needed on RBC and branchial CA activities in a range of elasmobranch species. Factors favoring plasma HCO3 − dehydration at the gill also exist for Pacific hagfish. Most notably, hagfish RBCs lack functional anion exchangers (Peters et al., 2000; Esbaugh et al., 2009), and this characteristic, together with low RBC CA activity (Maren et al., 1980; Esbaugh et al., 2009) and low hemoglobin buffering capacity and Haldane effect (Nikinmaa, 1997), are likely to limit the contribution of RBC HCO3 − dehydration to CO2 excretion. Branchial type IV-like CA and relatively high plasma buffering capacity will favor HCO3 − dehydration in the plasma, and indeed, Pacific hagfish carry the majority of the blood total CO2 load in the plasma (Esbaugh et al., 2009). Measurements of CO2 excretion before and after selective inhibition of extracellular CA activity are, however, needed to verify this model.

3. New insights into the role of branchial CA in ionic and acid–base regulation Although there is strong support for the view that branchial CA contributes to ionic and acid–base regulation, our understanding of the CA isoforms present in the gill, as well as their cellular and subcellular distribution, regulation, and specific roles, remains far from complete. The complexity of the gill – its architecture, diversity of cell types, and multifaceted responses to salinity and acid–base disturbances – has created significant challenges. For example, current models of ionic and acid–base regulation by the rainbow trout gill postulate the involvement of two ionocytes, a base-secreting cell that takes up Cl− (the peanut lectin agglutinin-positive mitochondrion-rich cell or PNA+ MR cell), and an acid-secreting cell that takes up Na+ (PNA− MR cell) (Goss et al., 2001; Galvez et al., 2002; see Evans et al., 2005; Perry and Gilmour, 2006; Tresguerres et al., 2006a). Compensation of acid–base disturbances involves different responses by each of these ionocytes; a metabolic acidosis is corrected by increasing Na+ /H+ exchange with little or no contribution of reduced Cl− /HCO3 − exchange (Goss

225

and Wood, 1991), whereas the opposite is true of a respiratory acidosis, where net acid excretion is driven primarily by reduced Cl− /HCO3 − exchange with a smaller contribution of enhanced Na+ /H+ exchange (Wood et al., 1984; Perry et al., 1987; Goss and Wood, 1990a). On the other hand, during correction of a metabolic alkalosis Cl− /HCO3 − exchange is enhanced (Goss and Wood, 1990b, 1991; Goss and Perry, 1994). Cytosolic CA is presumed to be present in both ionocyte types, catalyzing the hydration of CO2 to provide the acid–base equivalents for the ion transport mechanisms (Gilmour and Perry, 2009), and therefore responses of branchial CA to acid–base disturbances would be expected to be cell-type specific. This issue may explain why changes in CA mRNA abundance, protein levels and/or activity measured on gill homogenates from trout exposed to acid–base disturbances were not always in the direction predicted (e.g. Georgalis et al., 2006; Gilmour et al., 2011). A model that is gaining rapidly in popularity because it overcomes some of these difficulties is the skin of the developing zebrafish, Danio rerio. The zebrafish is in general an attractive model because the genetics information and approaches that are available for this species allow not only the functional analysis of specific genes, but also provide tools to knock down gene function (reviewed by Ekker and Akimenko, 2010). Prior to the formation of gills, the skin serves as the site of ionic and acid–base regulation, expressing a complement of ionocytes and responding to ionoregulatory and acid–base challenges (Rombough, 2004, 2007); indeed, it has been suggested that the basic physiology of ionic regulation is virtually identical in larval versus adult fish with the exception of location, i.e. skin versus gills (Rombough, 2007). Larval skin ionocytes are much more accessible than those of the adult gill, allowing techniques such as scanning ion-selective electrodes (SIET) to be used to assess ionocyte function in vivo. In larval zebrafish skin, three ionocyte types have been identified (reviewed by Hwang and Lee, 2007; Hwang, 2009; Hwang and Perry, 2010; Hwang et al., 2011): H+ -ATPase-rich (HR) cells that are involved in Na+ uptake and acid secretion (Lin et al., 2006; Esaki et al., 2007); Na+ ,K+ -ATPase-rich (NaR) cells that take up Ca2+ (Pan et al., 2005); and Na+ ,Cl− co-transporter (NCC) cells that are responsible for Cl− uptake and also contribute to Na+ uptake (Wang et al., 2009). To date, CA has been localized only to one of these ionocytes, the HR cell, where two isoforms were identified (Lin et al., 2008). The HR cell expresses a cytosolic CA isoform (variously termed zCA2-like a or zCAc) that is widely distributed in zebrafish tissues but distinct from the cytosolic isoform found in blood (Esaki et al., 2007; Lin et al., 2008). The HR cell also expresses a CA IV-like isoform, zCA15a (Lin et al., 2008). For both zCAc and zCA15a, localization to the HR cell has been at the level of mRNA (Lin et al., 2008); protein localization awaits the development of appropriate antibodies. The sequence similarity of zCA15a to mammalian and piscine CA IV genes, however, suggests that zCA15a should be membraneanchored via a GPI linkage and would therefore be expected to occur on the apical membrane of the HR cell, exposed to the surrounding water. Evidence implicating HR cell-CA in Na+ uptake as well as H+ excretion has come from studies using CA inhibitors and CA isoform knock down. Treatment of zebrafish larvae with ethoxzolamide, a membrane permeant CA inhibitor, reduced Na+ accumulation by HR cells although interestingly, without significantly reducing whole-body Na+ influx (Esaki et al., 2007). Knock down of either zCAc or zCA15a significantly increased whole-body Na+ influx, an unexpected effect that was attributed to compensatory responses favoring Na+ uptake by Na+ /H+ exchange over proton excretion by H+ -ATPase (Lin et al., 2008). Using a H+ -selective microelectrode, knock down of zCAc was found to significantly reduce proton excretion from the yolk sac surface of live larvae (Lin et al., 2008). The effects of zCA15a knock down, on the other hand, were time dependent. Proton excretion was significantly increased in 24 hpf

226

K.M. Gilmour / Respiratory Physiology & Neurobiology 184 (2012) 223–230

Fig. 2. A schematic representation of cell types in the zebrafish (Danio rerio) larval skin and/or gill epithelium that are thought to be responsible for (A) Na+ uptake linked to acid excretion (the HR cell, see Hwang, 2009; Hwang et al., 2011), and (B) Cl− uptake linked to base excretion (Bayaa et al., 2009). The role of cytosolic CA in both cases is to catalyze the hydration of CO2 to provide H+ and HCO3 − for export across the apical or basolateral membrane, as appropriate. The HR cell also expresses a type IV CA called CA15a, the role of which is at present unclear. Two possibilities are presented; CA15a may catalyze CO2 hydration to provide H+ for the formation of NH4 + (Wright and Wood, 2009), or it may catalyze the dehydration of water HCO3 − to provide CO2 that is recycled into the cell and hydrated (Hwang, 2009; Hwang and Perry, 2010; Hwang et al., 2011). Electroneutral exchangers are drawn as open circles while ATPases are drawn as filled circles. NHE3b, Na+ /H+ exchanger 3b; Rhcg1, Rh glycoprotein c1; CA15a, a type IV carbonic anhydrase isoform; CAc, cytosolic carbonic anhydrase, also termed CA II-like a; AE1, anion exchanger 1b; A3, SLC26A3 anion exchanger.

(hours post fertilization) embryos, but reduced in 96 hpf larvae, with the changes being attributed to the initiation of compensatory responses, as knock down of either CA isoform resulted in significant changes in the mRNA abundance of H+ -ATPase and the Na+ /H+ exchanger zNHE3b (Lin et al., 2008). The observation that zCA15a mRNA abundance increases in acidic water as well as low Na+ water, conditions that increase the difficulty of Na+ uptake and/or may cause acidosis, also is consistent with a role of zCA15a in Na+ uptake and/or H+ excretion (Lin et al., 2008). Collectively, these data support roles for both cytosolic CA (zCAc) and apical, membrane-bound CA (zCA15a) in a mechanism of Na+ uptake coupled to proton excretion within the HR cells of larval zebrafish skin (Fig. 2A). The role proposed for zCAc in this mechanism is to catalyze the hydration of CO2 within the HR cell to provide protons for export across the apical membrane; HCO3 − ions would be transferred back into plasma across the basolateral membrane (see Hwang, 2009; Hwang and Perry, 2010; Hwang et al., 2011). The role of zCA15a is less certain. By analogy with models of Na+ reabsorption at the mammalian renal proximal tubule

(Hamm and Nakhoul, 2008; Hamm et al., 2008), Hwang and colleagues proposed that zCA15a catalyzes the dehydration of water HCO3 − in the boundary layer next to the HR cell, with the protons for this dehydration reaction being supplied by H+ -ATPase and/or Na+ /H+ exchange (zNHE3b) (Hwang, 2009; Hwang et al., 2011). An alternative and perhaps more attractive possibility, however, stems from recent updates to models proposing a linkage between Na+ uptake and ammonia excretion (Wright and Wood, 2009). In these models (Fig. 2A), the apparently unfavorable gradients for the operation of apical Na+ /H+ exchange (Avella and Bornancin, 1989; reviewed by Parks et al., 2008) are overcome by co-localization of Na+ /H+ exchangers with Rh glycoproteins to create a ‘metabolon’. Movement of ammonia as NH3 via Rh glycoproteins would both provide protons for Na+ /H+ exchange intracellularly, as H+ is removed from NH4 + , and consume protons extracellularly, as NH4 + is formed, thereby providing a H+ gradient that would facilitate Na+ /H+ exchange. In this view, apical membrane-bound CA would catalyze the hydration of excreted CO2 , providing an additional source of protons for NH4 + formation (Wright and Wood, 2009). Several observations support this model of Na+ uptake for the zebrafish HR cell. The Rh glycoprotein Rhcg1 was colocalized with zNHE3b in HR cells (Nakada et al., 2007). Ammonia excretion was reduced by Rhcg1 knock down, demonstrating that this Rh glycoprotein provides an important route for ammonia excretion (Shih et al., 2008; Braun et al., 2009). Uptake of Na+ by Na+ /H+ exchange is thought to increase in importance in acidic water or water of low Na+ content, and under these conditions, Rhcg1 knock down decreased Na+ uptake (Kumai and Perry, 2011; Shih et al., 2012). While evidence in support of the NHE/Rh ‘metabolon’ model of Na+ uptake is accumulating, as yet there are insufficient data to determine whether zCA15a contributes to acidtrapping by catalyzing CO2 hydration (Wright and Wood, 2009), or proton removal by catalyzing HCO3 − dehydration (Hwang, 2009; Hwang et al., 2011). Carbonic anhydrase also contributes to Cl− uptake, at least in adult zebrafish, where treatment with ethoxzolamide nearly eliminated Cl− uptake (Boisen et al., 2003). The cellular location and mechanism of CA-dependent Cl− uptake in larval zebrafish have yet to be fully elucidated. Chloride uptake via the NCC cell relies on an apical Na+ ,Cl− co-transporter and does not require CA (Wang et al., 2009). Members of the SLC26 family of anion transporters have also been implicated in Cl− uptake by larval zebrafish (Bayaa et al., 2009). Knock down of SLC26A3 significantly reduced both Cl− uptake and base excretion, demonstrating a role for SLC26A3 Cl− /HCO3 − exchangers in Cl− uptake (Bayaa et al., 2009). Cells expressing SLC26A3 transcripts or protein were identified in the developing gill but not the skin, and were in some cases enriched in Na+ ,K+ -ATPase (Bayaa et al., 2009). These findings suggest the presence in the developing gill specifically of an ionocyte type that expresses SLC26A3 Cl− /HCO3 − exchangers and contributes to Cl− uptake and base excretion (Fig. 2B). However, operation of an electroneutral Cl− /HCO3 − exchanger for Cl− uptake from dilute freshwater is thermodynamically unfavorable unless mechanisms are in place to create local gradients that drive the exchanger (Tresguerres et al., 2006a). One such mechanism posits a basolateral proton pump, brought in close physical proximity to the apical anion exchanger via basolateral membrane infoldings, with cytosolic CA to catalyze the hydration of CO2 for the provision of H+ and HCO3 − (Tresguerres et al., 2006a). With this scenario, which would be consistent with data implicating CA in Cl− uptake (Boisen et al., 2003), SLC26A3-positive cells would also be expected to express cytosolic CA. This possibility has yet to be investigated. The use of larval zebrafish skin as a surrogate for the adult fish gill clearly has advanced, and will continue to advance, our understanding of mechanisms of ion uptake and acid–base excretion in freshwater fish. But how well does larval zebrafish skin actually

K.M. Gilmour / Respiratory Physiology & Neurobiology 184 (2012) 223–230

4. New insights into the role of branchial CA in CO2 /pH sensing The gill is widely accepted as a key site of both O2 and CO2 sensing in fish (see reviews by Gilmour, 2001; Milsom, 2002; Perry and Gilmour, 2002; Burleson and Milsom, 2003; Gilmour and Perry, 2007; Jonz and Nurse, 2008; Perry et al., 2009), with recent work suggesting that it also plays a role in sensing ammonia (Zhang and Wood, 2009; Zhang et al., 2011). The activation of gill chemoreceptors by changes in water or blood gas tensions is linked to the initiation of ventilatory and cardiovascular responses that attempt to minimize the deviation of arterial blood gases from normal set points. For example, exposure to hypoxia typically elicits hyperventilation, in an attempt to defend arterial O2 tension, and bradycardia (reviewed by Perry et al., 2009; Gamperl and Driedzic, 2009), while hyperventilatory responses to hypercapnia likely aim to minimize the extent of the (unavoidable) increase in arterial CO2 tension and its attendant acidosis (Gilmour, 2001). The branchial cell type implicated in O2 - (Jonz et al., 2004; Burleson et al., 2006), CO2 (Qin et al., 2010), and NH3 -sensing (Zhang et al., 2011) is the neuroepithelial cell (NEC; Dunel-Erb et al., 1982). Recent work suggests that zebrafish NECs express CA that contributes to CO2 sensing (Qin et al., 2010). Neuroepithelial cells, identified on the basis of positive immunoreactivity for serotonin (Dunel-Erb et al., 1982; Jonz and Nurse, 2003), were also immunopositive for CA (Qin et al., 2010). Further work is required to characterize the specific CA isoform involved, although it is presumed to be cytosolic (likely zCAc) based on size (29 kDa) and the positive response obtained with antibodies against trout cytosolic CA (tCAc; Georgalis et al., 2006) and human CA II (Qin et al., 2010). Using whole-cell patch-clamp techniques, Qin et al. (2010) demonstrated that the depolarization response of NECs to CO2 (under conditions of constant pH) was inhibited by acetazolamide; the magnitude of membrane depolarization was reduced and the time required to achieve maximal response was longer. Models of CO2 sensing by glomus cells of the mammalian carotid body postulate a role for CA in catalyzing the hydration of CO2 to protons, which then inhibit a background or leak K+ channel to cause depolarization of the cell (Iturriaga et al., 1991; Buckler

CO2 Water HCO-3

CAc

CO2

..... ..... 2+ . Ca ... ... ..... ..... .........

H ↓

mimic the adult zebrafish gill? There are surprisingly few empirical data available to address this question. Given the power and popularity of this model, the need for direct comparisons of the ionocyte complements of larval zebrafish skin and adult zebrafish gill is becoming increasingly urgent. Applying knowledge gained from studies of zebrafish to describing the ionocytes and cellular mechanisms of ionic and acid–base regulation in other fish species is another obvious, necessary and yet very challenging next step. To date, progress has been made with only a handful of species, including rainbow trout (Goss et al., 2001; Galvez et al., 2002), tilapia (Oreochromis mossambicus, Hiroi et al., 2005; Inokuchi et al., 2009) and medaka (Oryzias latipes, Wu et al., 2010; Lin et al., 2012). Within (and beyond) these model species, the role of CA in ion uptake and acid–base regulatory mechanisms clearly requires further study, particularly with respect to identifying the CA isoforms that are present in larval skin and/or adult gill, as well as their cellular and subcellular localization. The use of euryhaline fish for such studies would be particularly interesting, because CA is not implicated in cellular mechanisms of NaCl secretion at the marine teleost gill, but is expected to be involved in branchial mechanisms of acid–base excretion in marine teleosts as well as elasmobranch and agnathan fish (see reviews by Claiborne et al., 2002; Evans et al., 2005; Hwang et al., 2011). Despite this expectation, few studies have examined the role of branchial CA in acid–base excretion in marine fish (Swenson and Maren, 1987; Mallatt et al., 1987; Maren et al., 1992; Wilson et al., 2000; Tresguerres et al., 2007).

227

+

H

Gill epithelium

Blood

K+ Fig. 3. A schematic representation of CO2 detection by CO2 -sensitive neuroepithelial cells of the zebrafish gill (Qin et al., 2010). Cytosolic CA catalyzes the hydration of CO2 to provide H+ that inhibits a background K+ channel, resulting in cellular depolarization. Depolarization is presumed to be followed by Ca2+ -dependent neurosecretion, although these elements of the signal transduction pathway have yet to be demonstrated experimentally. CAc, cytosolic carbonic anhydrase.

et al., 1991; Iturriaga, 1993; Iturriaga and Lahiri, 1993; Gonzalez et al., 1994) and the results for zebrafish NECs are consistent with this model (Fig. 3; Qin et al., 2010), although additional work is needed to characterize the CO2 -sensing mechanism of fish NECs more completely (see also Perry and Abdallah, in press). A role has also been proposed for CA in pH sensing by the fish gill (Tresguerres et al., 2007). The pH sensitivity of NECs (Qin et al., 2010) and whether fish alter ventilation in response to changes of systemic pH remain open questions (reviewed by Gilmour, 2001; Gilmour and Perry, 2007). Theoretical considerations suggest that the capacity of fish to adjust acid–base status by modulating ventilation is very limited (Heisler, 1986; Gilmour, 2001; Claiborne et al., 2002), and experimental tests of CO2 versus pH as ventilatory stimuli have identified changes in water CO2 tension as the major if not sole trigger of cardiorespiratory reflexes (e.g. Perry and McKendry, 2001; Perry and Reid, 2002; Gilmour et al., 2005). Nevertheless, several studies have documented changes in ventilation that track arterial pH rather than arterial CO2 tension (Heisler et al., 1988; Graham et al., 1990; Wood et al., 1990; Wood and Munger, 1994), leaving open the possibility that acid–base disturbances may stimulate ventilatory responses. Metabolic compensation, however, remains the key response to acid–base disturbances, with the gill as the dominant effector of acid–base compensation and the kidney playing a supporting role (Perry and Gilmour, 2006). Despite widespread agreement on this point, our knowledge of how acid–base disturbances are detected and metabolic compensation is initiated remains scanty. In dogfish subjected to metabolic alkalosis by base infusion, a key compensatory response involves H+ -ATPase translocation to the basolateral membrane of cells that are thought to excrete base across the apical membrane (Tresguerres et al., 2005, 2006b). The response is eliminated by treatment of fish with acetazolamide (Tresguerres et al., 2007), implicating CA in the sensory mechanism that detects alkalosis and initiates H+ -ATPase translocation. The response is also eliminated in fish treated with inhibitors of soluble adenylyl cyclase (sAC), a cytosolic (not membrane-associated), cAMP-producing enzyme that is activated by HCO3 − (Tresguerres et al., 2010). Collectively, these findings suggest a sensory mechanism in which CA-catalyzed production of HCO3 − activates sAC. The base-sensing mechanism appears to be located in the gill itself, because H+ -ATPase translocation can be stimulated by perfusing isolated gills with alkaline saline (Tresguerres et al., 2010). Tresguerres et al. (2010) integrated these findings into a model in which HCO3 − enters the cell as CO2 following dehydration in the plasma catalyzed by pillar cell CA IV (Fig. 1). Once in the cell,

228

K.M. Gilmour / Respiratory Physiology & Neurobiology 184 (2012) 223–230

CO2 is hydrated to H+ and HCO3 − in the presence of cytosolic CA, with HCO3 − being exported across the apical membrane by a pendrin-type anion exchanger (SLC26A4) and H+ exiting the cell via basolateral H+ -ATPase. Cytosolic CA and H+ -ATPase appear to be co-localized in dogfish gills (Tresguerres et al., 2007), and a pendrintype anion exchanger and H+ -ATPase were co-localized to the same cell in the gill of a different elasmobranch, the Atlantic stingray Dasyatis sabina (Piermarini et al., 2002). During metabolic alkalosis, increased HCO3 − entry into the cell (as CO2 ) is proposed to activate sAC, stimulating translocation of H+ -ATPase to the basolateral membrane to increase the capacity for base excretion (Tresguerres et al., 2010). A number of questions remain to be resolved with this model for the detection of alkaline pH and resultant initiation of H+ -ATPase translocation. For example, it is not clear how different responses to metabolic alkalosis (elevated plasma HCO3 − requiring base excretion) and respiratory acidosis (elevated plasma PCO2 and H+ requiring acid excretion) would be obtained if the stimulus for sAC activation is HCO3 − derived from CO2 hydration. Similar questions remain to be resolved for the clear cells of the mammalian epididymis and the A- and B-type intercalated cells of the mammalian kidney, other locations in which comparable, cellular models of acid–base sensing have been proposed (and tested at least to some degree) owing to the co-occurrence of sAC, cytosolic CA, expression on opposite membranes of H+ -ATPase and anion exchangers, and involvement of H+ -ATPase translocation in response to acid–base challenges (Pastor-Soler et al., 2003; Brown et al., 2009; Shum et al., 2009). Importantly, the work of Tresguerres et al. (2010) suggests that regulation of systemic pH results from the detection of acid–base disturbances by the cells that initiate appropriate compensatory mechanisms, i.e. that compensatory responses can be initiated without a specialized sensory cell that acts through the nervous and/or endocrine systems to coordinate the excretion of acid–base molecules at gill and kidney. With this point in mind, it will be important to investigate cellular mechanisms of acid–base detection at the kidney as well, of course, as extending such studies to other fish species.

5. Perspectives The application of molecular techniques to identify CA isoforms in conjunction with the use of powerful models (e.g. larval fish, isolated cell preparations) has provided new insights into the localization and function of CA in the fish gill. This work has revealed that fish utilize more than one model of CO2 excretion, that to understand the role of CA and the gill in ionic regulation and acid–base balance means characterizing the transporter and CA complement of individual cell types, and that CA plays roles in branchial sensory mechanisms. In many cases the advances have been made by focusing on a particular species, and clearly much work is needed to identify comparable (or different!) mechanisms in a range of species. The regulation of CA expression and activity is also an area that warrants investigation. A number of studies have documented changes in branchial CA mRNA abundance, protein levels and/or activity with salinity changes (e.g. Perry and Laurent, 1990; Kültz et al., 1992; Chang and Hwang, 2004; Scott et al., 2005, 2008; Craig et al., 2007; Lin et al., 2008) or acid–base challenges (e.g. Dimberg and Höglund, 1987; Hirata et al., 2003; Georgalis et al., 2006; Lin et al., 2008; Gilmour et al., 2011), but virtually nothing is known of the mechanisms through which these changes are achieved. The fish gill is a dynamic and multi-functional organ (Evans et al., 2005) of abundant CA expression. Given that the reaction catalyzed by CA is fundamental to an array of physiological processes that occur at the gill, the study of branchial CA is important both in understanding gill function and in understanding the diversity and regulation of this key enzyme.

Acknowledgment Original research presented in this paper was supported by NSERC of Canada Discovery and Research Tools & Instruments grants.

References Avella, M., Bornancin, M., 1989. A new analysis of ammonia and sodium transport through the gills of the freshwater rainbow trout (Salmo gairdneri). Journal of Experimental Biology 142, 155–175. Bayaa, M., Vulesevic, B., Esbaugh, A., Braun, M., Ekker, M., Grosell, M., Perry, S.F., 2009. The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio) larvae. Journal of Experimental Biology 212, 3283–3295. Berenbrink, M., Koldkjær, P., Kepp, O., Cossins, A.R., 2005. Evolution of oxygen secretion in fishes and the emergence of a complex physiological system. Science 307, 1752–1757. Boisen, A.M.Z., Amstrup, J., Novak, I., Grosell, M., 2003. Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio). Biochimica et Biophysica Acta 1618, 207–218. Braun, M.H., Steele, S.L., Ekker, M., Perry, S.F., 2009. Nitrogen excretion in developing zebrafish (Danio rerio): a role for Rh proteins and urea transporters. American Journal of Physiology 296, F994–F1005. Brown, D., Paunescu, T.G., Breton, S., Marshansky, V., 2009. Regulation of the VATPase in kidney epithelial cells: dual role in acid–base homeostasis and vesicle trafficking. Journal of Experimental Biology 212, 1762–1772. Buckler, K.J., Vaughan-Jones, R.D., Peers, C., Nye, P.C.G., 1991. Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat. The Journal of Physiology 436, 107–129. Burleson, M.L., Mercer, S.E., Wilk-Blaszczak, M.A., 2006. Isolation and characterization of putative O2 chemoreceptor cells from the gills of channel catfish (Ictalurus punctatus). Brain Research 1092, 100–107. Burleson, M.L., Milsom, W.K., 2003. Comparative aspects of O2 chemoreception. Anatomy, physiology, and environmental applications. In: Lahiri, S., Semenza, G.L., Prabhakar, N.R. (Eds.), Oxygen Sensing. Responses and Adaptation to Hypoxia. Marcel Dekker, New York, pp. 685–707. Butler, P.J., Metcalfe, J.D., 1988. Cardiovascular and respiratory systems. In: Shuttleworth, T.J. (Ed.), Physiology of Elasmobranch Fishes. Springer-Verlag, Berlin, pp. 1–47. Chang, I.-C., Hwang, P.-P., 2004. Cl− uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus). Physiological and Biochemical Zoology 77, 406–414. Claiborne, J.B., Edwards, S.L., Morrison-Shetlar, A.I., 2002. Acid–base regulation in fishes: cellular and molecular mechanisms. Journal of Experimental Zoology 293, 302–319. Craig, P.M., Wood, C.M., McLelland, G.B., 2007. Gill membrane remodeling with softwater acclimation in zebrafish (Danio rerio). Physiological Genomics 30, 53–60. Dimberg, K., Höglund, L.B., 1987. Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard, bicarbonate-rich freshwater. Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology 157, 405–412. Dunel-Erb, S., Bailly, Y., Laurent, P., 1982. Neuroepithelial cells in fish gill primary lamellae. Journal of Applied Physiology 53, 1342–1353. Ekker, M., Akimenko, M.-A., 2010. Genetic tools. In: Perry, S.F., Ekker, M., Farrell, A.P., Brauner, C.J. (Eds.), Fish Physiology v29 Zebrafish. Academic Press, San Diego, pp. 1–23. Esaki, M., Hoshijima, K., Kobayashi, S., Fukuda, H., Kawakami, K., Hirose, S., 2007. Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a. American Journal of Physiology 292, R470–R480. Esbaugh, A., Tufts, B.L., 2006. The structure and function of carbonic anhydrase isozymes in the respiratory system of vertebrates. Respiratory Physiology and Neurobiology 154, 185–198. Esbaugh, A.J., Gilmour, K.M., Perry, S.F., 2009. Membrane-associated carbonic anhydrase in the respiratory system of the Pacific hagfish (Eptatretus stouti). Respiratory Physiology and Neurobiology 166, 107–116. Evans, D.H., Piermarini, P.M., Choe, K.P., 2005. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid–base regulation, and excretion of nitrogenous waste. Physiological Reviews 85, 97–177. Galvez, F., Reid, S.D., Hawkings, G.S., Goss, G.G., 2002. Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout. American Journal of Physiology 282, R658–R668. Gamperl, A.K., Driedzic, W.R., 2009. Cardiovascular function and cardiac metabolism. In: Richards, J.G., Farrell, A.P., Brauner, C.J. (Eds.), Fish Physiology v27 Hypoxia. Academic Press, San Diego, pp. 301–360. Georgalis, T., Perry, S.F., Gilmour, K.M., 2006. The role of branchial carbonic anhydrase in acid–base regulation in rainbow trout (Oncorhynchus mykiss). Journal of Experimental Biology 209, 518–530. Gilmour, K.M., 2001. The CO2 /pH ventilatory drive in fish. Comparative Biochemistry and Physiology. A: Comparative Physiology 130, 219–240. Gilmour, K.M., Bayaa, M., Kenney, L., McNeill, B., Perry, S.F., 2007. Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias). American Journal of Physiology 292, R556–R567.

K.M. Gilmour / Respiratory Physiology & Neurobiology 184 (2012) 223–230 Gilmour, K.M., Collier, C.L., Dey, C.J., Perry, S.F., 2011. Roles of cortisol and carbonic anhydrase in acid–base compensation in rainbow trout, Oncorhynchus mykiss. Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology 181, 501–515. Gilmour, K.M., Henry, R.P., Wood, C.M., Perry, S.F., 1997. Extracellular carbonic anhydrase and an acid–base disequilibrium in the blood of the dogfish, Squalus acanthias. Journal of Experimental Biology 200, 173–183. Gilmour, K.M., Milsom, W.K., Rantin, F.T., Reid, S.G., Perry, S.F., 2005. Cardiorespiratory responses to hypercarbia in tambaqui (Colossoma macropomum): chemoreceptor orientation and specificity. Journal of Experimental Biology 208, 1095–1107. Gilmour, K.M., Perry, S.F., 2004. Branchial membrane-associated carbonic anhydrase activity maintains CO2 excretion in severely anemic dogfish. American Journal of Physiology 286, R1138–R1148. Gilmour, K.M., Perry, S.F., 2007. Branchial chemoreceptor regulation of cardiorespiratory function. In: Zielinski, B., Hara, T.J. (Eds.), Fish Physiology v 25 Sensory Systems Neuroscience. Academic, San Diego, pp. 97–151. Gilmour, K.M., Perry, S.F., 2009. Carbonic anhydrase and acid–base regulation in fish. Journal of Experimental Biology 212, 1647–1661. Gilmour, K.M., Perry, S.F., 2010. Gas transfer in dogfish: a unique model of CO2 excretion. Comparative Biochemistry and Physiology. A: Comparative Physiology 155, 476–485. Gilmour, K.M., Perry, S.F., Bernier, N.J., Henry, R.P., Wood, C.M., 2001. Extracellular carbonic anhydrase in dogfish, Squalus acanthias: a role in CO2 excretion. Physiological and Biochemical Zoology 74, 477–492. Gilmour, K.M., Shah, B., Szebedinszky, C., 2002. An investigation of carbonic anhydrase activity in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate (Raja rhina), and spotted ratfish (Hydrolagus colliei). Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology 172, 77–86. Gonzalez, C., Almaraz, L., Obeso, A., Rigual, R., 1994. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiological Reviews 74, 829–898. Goss, G.G., Adamia, S., Galvez, F., 2001. Peanut lectin binds to a subpopulation of mitochondria-rich cells in the rainbow trout gill epithelium. American Journal of Physiology 281, R1718–R1725. Goss, G.G., Perry, S.F., 1994. Different mechanisms of acid–base regulation in rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) during NaHCO3 infusion. Physiological Zoology 67, 381–406. Goss, G.G., Wood, C.M., 1990a. Na+ and Cl− uptake kinetics, diffusive effluxes and acidic equivalent fluxes across the gills of rainbow trout. I. Responses to environmental hyperoxia. Journal of Experimental Biology 152, 521–547. Goss, G.G., Wood, C.M., 1990b. Na+ and Cl− uptake kinetics, diffusive effluxes and acidic equivalent fluxes across the gills of rainbow trout. II. Responses to bicarbonate infusion. Journal of Experimental Biology 152, 549–571. Goss, G.G., Wood, C.M., 1991. Two-substrate kinetic analysis: a novel approach linking ion and acid–base transport at the gills of freshwater trout, Oncorhynchus mykiss. Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology 161, 635–646. Graham, M.S., Turner, J.D., Wood, C.M., 1990. Control of ventilation in the hypercapnic skate Raja ocellata: I. blood and extradural fluid. Respiration Physiology 80, 259–277. Hamm, L.L., Alpern, R.J., Preisig, P.A., 2008. Cellular mechanisms of renal tubular acidification. In: Alpern, R.J., Hebert, S.C. (Eds.), Seldin and Giebisch’s The Kidney. Physiology and Pathophysiology. Academic Elsevier, Amsterdam, pp. 1539–1585. Hamm, L.L., Nakhoul, N.L., 2008. Renal acidifcation. In: Brenner, B.M. (Ed.), Brenner & Rector’s The Kidney. , 8th ed. Saunders Elsevier, Philadelphia, pp. 248–279. Haswell, M.S., Randall, D.J., Perry, S.F., 1980. Fish gill carbonic anhydrase: acid–base regulation or salt transport? American Journal of Physiology 238, R240–R245. Heisler, N., 1986. Comparative aspects of acid–base regulation. In: Heisler, N. (Ed.), Acid–Base Regulation in Animals. Elsevier, Amsterdam, pp. 397–449. Heisler, N., Toews, D.P., Holeton, G.F., 1988. Regulation of ventilation and acid–base status in the elasmobranch Scyliorhinus stellaris during hyperoxia-induced hypercapnia. Respiration Physiology 71, 227–246. Henry, R.P., Gilmour, K.M., Wood, C.M., Perry, S.F., 1997. Extracellular carbonic anhydrase activity and carbonic anhydrase inhibitors in the circulatory system of fish. Physiological Zoology 70, 650–659. Henry, R.P., Heming, T.A., 1998. Carbonic anhydrase and respiratory gas exchange. In: Perry, S.F., Tufts, B.L. (Eds.), Fish Respiration. Academic Press, San Diego, pp. 75–111. Henry, R.P., Swenson, E.R., 2000. The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Respiration Physiology 121, 1–12. Hilvo, M., Tolvanen, M., Clark, A., Shen, B., Shah, G.N., Waheed, A., Halmi, P., Hänninen, M., Hämäläinen, J.M., Vihinen, M., Sly, W.S., Parkkila, S., 2005. Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase. Biochemical Journal 392, 83–92. Hirata, T., Kaneko, T., Ono, T., Nakazato, T., Furukawa, N., Hasegawa, S., Wakabayashi, S., Shigekawa, M., Chang, M.-H., Romero, M.F., Hirose, S., 2003. Mechanism of acid adaptation of a fish living in a pH 3.5 lake. American Journal of Physiology 284, R1199–R1212. Hiroi, J., McCormick, S.D., Ohtani-Kaneko, R., Kaneko, T., 2005. Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+ /K+ -ATPase, Na+ /K+ /2Cl− cotransporter and CFTR anion channel. Journal of Experimental Biology 208, 2023–2036.

229

Hwang, P.-P., 2009. Ion uptake and acid secretion in zebrafish (Danio rerio). Journal of Experimental Biology 212, 1745–1752. Hwang, P.-P., Lee, T.-H., 2007. New insights into fish ion regulation and mitochondrion-rich cells. Comparative Biochemistry and Physiology. A: Comparative Physiology 148, 479–497. Hwang, P.-P., Lee, T.-H., Lin, L.-Y., 2011. Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. American Journal of Physiology 301, R28–R47. Hwang, P.-P., Perry, S.F., 2010. Ionic and acid–base regulation. In: Perry, S.F., Ekker, M., Farrell, A.P., Brauner, C.J. (Eds.), Fish Physiology v29 Zebrafish. Academic, San Diego, pp. 311–344. Inokuchi, M., Hiroi, J., Watanabe, S., Hwang, P.-P., Kaneko, T., 2009. Morphological and functional classification of ion-absorbing mitochondria-rich cells in the gills of Mozambique tilapia. Journal of Experimental Biology 212, 1003–1010. Iturriaga, R., 1993. Carotid body chemoreception: the importance of CO2 –HCO3 − and carbonic anhydrase. Biological Research 26, 319–329. Iturriaga, R., Lahiri, S., 1993. Carbonic anhydrase and carotid body chemoreception in the presence and absence of CO2 –HCO3 − . Advances in Experimental Medicine and Biology 337, 171–176. Iturriaga, R., Lahiri, S., Mokashi, A., 1991. Carbonic anhydrase and chemoreception in the cat carotid body. American Journal of Physiology 261, C565–C573. Jonz, M.G., Fearon, I.M., Nurse, C.A., 2004. Neuroepithelial oxygen chemoreceptors of the zebrafish gill. The Journal of Physiology 560, 737–752. Jonz, M.G., Nurse, C.A., 2003. Neuroepithelial cells and associated innervation of the zebrafish gill: a confocal immunofluorescence study. The Journal of Comparative Neurology 461, 1–17. Jonz, M.G., Nurse, C.A., 2008. New developments on gill innervation: insights from a model vertebrate. Journal of Experimental Biology 211, 2371–2378. Kerstetter, T.H., Kirschner, L.B., 1972. Active chloride transport by the gills of rainbow trout (Salmo gairdneri). Journal of Experimental Biology 56, 263–272. Kültz, D., Bastrop, R., Jürss, K., Siebers, D., 1992. Mitochondria-rich (MR) cells and the activities of the Na+ /K+ -ATPase and carbonic anhydrase in the gill and opercular epithelium of Oreochromis mossambicus adapted to various salinities. Comparative Biochemistry and Physiology 102B, 293–301. Kumai, Y., Perry, S.F., 2011. Ammonia excretion via Rhcg1 facilitate Na+ uptake in larval zebrafish, Danio rerio, in acidic water. American Journal of Physiology 301, R1517–R1528. Lin, C.-C., Lin, L.-Y., Hsu, H.-H., Thermes, V., Prunet, P., Horng, J.-L., Hwang, P.-P., 2012. Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater. American Journal of Physiology 302, R283–R291. Lin, L.-Y., Horng, J.-L., Kunkel, J.G., Hwang, P.-P., 2006. Proton pump-rich cell secretes acid in skin of zebrafish larvae. American Journal of Physiology 290, C371–C378. Lin, T.-Y., Liao, B.-K., Horng, J.-L., Yah, J.-J., Hsiao, C.-D., Hwang, P.-P., 2008. Carbonic anhydrase 2-like a and 15a are involved in acid–base regulation and Na+ uptake in zebrafish H+ -ATPase-rich cells. American Journal of Physiology 294, C1250–C1260. Maetz, J., 1971. Fish gills: mechanisms of salt transfer in fresh water and sea water. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 262, 209–249. Maetz, J., Garcia-Romeu, F., 1964. The mechanism of sodium and chloride uptake by the gills of a fresh-water fish, Carassius auratus. II. Evidence for NH4 + /Na+ and HCO3 − /Cl− exchanges. Journal of General Physiology 47, 1209–1227. Mallatt, J., Conley, D.M., Ridgway, R.L., 1987. Why do hagfish have gill chloride cells when they need not regulate plasma NaCl concentration? Canadian Journal of Zoology 65, 1956–1965. Maren, T.H., 1967. Carbonic anhydrase: chemistry, physiology, and inhibition. Physiological Reviews 47, 595–781. Maren, T.H., Fine, A., Swenson, E.R., Rothman, D., 1992. Renal acid–base physiology in marine teleost, the long-horned sculpin (Myoxocephalus octodecimspinosus). American Journal of Physiology 263, F49–F55. Maren, T.H., Friedland, B.R., Rittmaster, R.S., 1980. Kinetic properties of primitive vertebrate carbonic anhydrases. Comparative Biochemistry and Physiology 67B, 69–74. Milsom, W.K., 2002. Phylogeny of CO2 /H+ chemoreception in vertebrates. Respiratory Physiology and Neurobiology 131, 29–41. Nakada, T., Hoshijima, K., Esaki, M., Nagayoshi, S., Kawakami, K., Hirose, S., 2007. Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression. American Journal of Physiology 293, R1743–R1753. Nikinmaa, M., 1997. Oxygen and carbon dioxide transport in vertebrate erythrocytes: an evoutionary change in the role of membrane transport. Journal of Experimental Biology 200, 369–380. Pan, T.-C., Liao, B.-K., Huang, C.-J., Lin, L.-Y., Hwang, P.-P., 2005. Epithelial Ca2+ channel expression and Ca2+ uptake in developing zebrafish. American Journal of Physiology 289, R1202–R1211. Parks, S.K., Tresguerres, M., Goss, G.G., 2008. Theoretical considerations underlying Na+ uptake mechanisms in freshwater fishes. Comparative Biochemistry and Physiology. C: Comparative Pharmacology 148, 411–418. Pastor-Soler, N., Beaulieu, V., Litvin, T.N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L.R., Breton, S., 2003. Bicarbonate-regulated adenylyl cylcase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. Journal of Biological Chemistry 278, 49523–49529. Payan, P., Matty, A.J., Maetz, J., 1975. A study of the sodium pump in the perfused head preparation of the trout Salmo gairdneri in freshwater. Journal of Comparative Physiology 104, 33–48.

230

K.M. Gilmour / Respiratory Physiology & Neurobiology 184 (2012) 223–230

Perry, S.F., 1986. Carbon dioxide excretion in fishes. Canadian Journal of Zoology 64, 565–572. Perry, S.F., Abdallah, S. Mechanisms and consequences of carbon dioxide sensing in fish. Respiratory Physiology and Neurobiology, http://dx.doi.org/10.1016/j.resp.2012.06.013, in press. Perry, S.F., Davie, P.S., Daxboeck, C., Randall, D.J., 1982. A comparison of CO2 excretion in a spontaneously ventilating blood-perfused trout preparation and salineperfused gill preparations: contribution of the branchial epithelium and red blood cell. Journal of Experimental Biology 101, 47–60. Perry, S.F., Gilmour, K.M., 2002. Sensing and transfer of respiratory gases at the fish gill. Journal of Experimental Zoology 293, 249–263. Perry, S.F., Gilmour, K.M., 2006. Acid–base balance and CO2 excretion in fish: unanswered questions and emerging models. Respiratory Physiology and Neurobiology 154, 199–215. Perry, S.F., Jonz, M.G., Gilmour, K.M., 2009. Oxygen sensing and the hypoxic ventilatory response. In: Richards, J.G., Farrell, A.P., Brauner, C.J. (Eds.), Fish Physiology v27 Hypoxia. Elsevier, Amsterdam, pp. 193–253. Perry, S.F., Laurent, P., 1990. The role of carbonic anhydrase in carbon dioxide excretion, acid–base balance and ionic regulation in aquatic gill breathers. In: Truchot, J.-P., Lahlou, B. (Eds.), Animal Nutrition and Transport Processes. 2. Transport, Respiration and Excretion: Comparative and Environmental Aspects. Karger, Basel, pp. 39–57. Perry, S.F., Malone, S., Ewing, D., 1987. Hypercapnic acidosis in the rainbow trout (Salmo gairdneri). I. Branchial ionic fluxes and blood acid–base status. Canadian Journal of Zoology 65, 888–895. Perry, S.F., McKendry, J.E., 2001. The relative roles of external and internal CO2 versus H+ in eliciting the cardiorespiratory responses of Salmo salar and Squalus acanthias to hpercarbia. Journal of Experimental Biology 204, 3963–3971. Perry, S.F., Payan, P., Girard, J.P., 1984. The effects of perfuate HCO3 − and PCO2 on chloride uptake in perfused gills of rainbow trout (Salmo gairdneri). Canadian Journal of Fisheries and Aquatic Sciences 41, 1768–1773. Perry, S.F., Reid, S.G., 2002. Cardiorespiratory adjustments during hypercarbia in rainbow trout Oncorhynchus mykiss are initiated by external CO2 receptors on the first gill arch. Journal of Experimental Biology 205, 3357–3365. Perry, S.F., Shahsavarani, A., Georgalis, T., Bayaa, M., Furimsky, M., Thomas, S., 2003. Channels, pumps, and exchangers in the gill and kidney of freshwater fishes: their role in ionic and acid–base regulation. Journal of Experimental Zoology 300A, 53–62. Perry, S.F., Wood, C.M., Walsh, P.J., Thomas, S., 1996. Fish red blood cell carbon dioxide transport in vitro: a comparative study. Comparative Biochemistry and Physiology 113A, 121–130. Peters, T., Forster, R.E., Gros, G., 2000. Hagfish (Myxine glutinosa) red cell membrane exhibits no bicarbonate permeability as detected by 18 O exchange. Journal of Experimental Biology 203, 1551–1560. Piermarini, P.M., Verlander, J.W., Royauz, I.E., Evans, D.H., 2002. Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch. American Journal of Physiology 283, R983–R992. Qin, Z., Lewis, J.E., Perry, S.F., 2010. Zebrafish (Danio rerio) gill neuroepithelial cells are sensitive chemoreceptors for environmental CO2 . The Journal of Physiology 588, 861–872. Rombough, P., 2007. The functional ontogeny of the teleost gill: which comes first, gas or ion exchange? Comparative Biochemistry and Physiology. A: Comparative Physiology 148, 732–742. Rombough, P.J., 2004. Gas exchange, ionoregulation, and the functional development of the teleost gill. American Fisheries Society Symposium 40, 47–83. Scott, G.R., Baker, D.W., Schulte, P.M., Wood, C.M., 2008. Physiological and molecular mechanisms of osmoregulatory plasticity in killifish after seawater transfer. Journal of Experimental Biology 211, 2450–2459. Scott, G.R., Claiborne, J.B., Edwards, S.L., Schulte, P.M., Wood, C.M., 2005. Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport. Journal of Experimental Biology 208, 2719–2729. Shih, T.-H., Horng, J.-L., Hwang, P.-P., Lin, L.-Y., 2008. Ammonia excretion by the skin of zebrafish (Danio rerio) larvae. American Journal of Physiology 295, C1625–C1632. Shih, T.-H., Horng, J.-L., Liu, S.-T., Hwang, P.-P., Lin, L.-Y., 2012. Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water. American Journal of Physiology 302, R84–R93.

Shum, W.W.C., Da Silva, N., Brown, D., Breton, S., 2009. Regulation of luminal acidification in the male reproductive tract via cell–cell crosstalk. Journal of Experimental Biology 212, 1753–1761. Sobotka, H., Kann, S., 1941. Carbonic anhydrase in fishes and invertebrates. Journal of Cellular and Comparative Physiology 17, 341–348. Supuran, C.T., Scozzafava, A., 2004. Benzolamide is not a membrane-impermeant carbonic anhydrase inhibitor. Journal of Enzyme Inhibition and Medicinal Chemistry 19, 269–273. Swenson, E.R., 2000. Respiratory and renal roles of carbonic anhydrase in gas exchange and acid–base regulation. EXS 90, 281–341. Swenson, E.R., Maren, T.H., 1987. Roles of gill and red cell carbonic anhydrase in elasmobranch HCO3 − and CO2 excretion. American Journal of Physiology 253, R450–R458. Tresguerres, M., Katoh, F., Fenton, H., Jasinska, E., Goss, G.G., 2005. Regulation of branchial V-H+ -ATPase, Na+ /K+ -ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias). Journal of Experimental Biology 208, 345–354. Tresguerres, M., Katoh, F., Orr, E., Parks, S.K., Goss, G.G., 2006a. Chloride uptake and base secretion in freshwater fish: a transepithelial ion-transport metabolon? Physiological and Biochemical Zoology 79, 981–996. Tresguerres, M., Parks, S.K., Katoh, F., Goss, G.G., 2006b. Microtubule-dependent relocation of branchial V-H+ -ATPase to the basolateral membrane in the Pacific spiny dogfish (Squalus acanthias): a role in base secretion. Journal of Experimental Biology 209, 599–609. Tresguerres, M., Parks, S.K., Salazar, E., Levin, L.R., Goss, G.G., Buck, J., 2010. Bicarbonate-sensing soluble adenylyl cyclase is an essential sensor for acid/base homeostasis. Proceedings of the National Academy of Sciences of the United States of America 107, 442–447. Tresguerres, M., Parks, S.K., Wood, C.M., Goss, G.G., 2007. V-H+ -ATPase translocation during blood alkalosis in dogfish gills: interaction with carbonic anhydrase and involvement in the postfeeding alkaline tide. American Journal of Physiology 292, R2012–R2019. Tufts, B.L., Perry, S.F., 1998. Carbon dioxide transport and excretion. In: Perry, S.F., Tufts, B.L. (Eds.), Fish Respiration. Academic Press, San Diego, pp. 229–281. Wang, Y.-F., Tseng, Y.-C., Yan, J.-J., Hiroi, J., Hwang, P.-P., 2009. Role of SLC12A10.2, a Na–Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio). American Journal of Physiology 296, R1650–R1660. Wilson, J.M., Randall, D.J., Donowitz, M., Vogl, A.W., Ip, A.K.Y., 2000. Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri). Journal of Experimental Biology 203, 2297–2310. Wood, C.M., Munger, R.S., 1994. Carbonic anhydrase injection provides evidence for the role of blood acid–base status in stimulating ventilation after exhaustive exercise in rainbow trout. Journal of Experimental Biology 194, 225–253. Wood, C.M., Perry, S.F., Walsh, P.J., Thomas, S., 1994. HCO3 − dehydration by the blood of an elasmobranch in the absence of a Haldane effect. Respiration Physiology 98, 319–337. Wood, C.M., Turner, J.D., Munger, R.S., Graham, M.S., 1990. Control of ventilation in the hypercapnic skate Raja ocellata: II. cerebrospinal fluid and intracellular pH in the brain and other tissues. Respiration Physiology 80, 279–298. Wood, C.M., Wheatly, M.G., Hõbe, H., 1984. The mechanisms of acid–base and ionoregulation in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia. III. Branchial exchanges. Respiration Physiology 55, 175–192. Wright, P.A., Wood, C.M., 2009. A new paradigm for ammonia excretion in aquatic animals: role of Rhesus (Rh) glycoproteins. Journal of Experimental Biology 212, 2303–2312. Wu, S.-C., Horng, J.-L., Liu, S.-T., Hwang, P.-P., Wen, Z.-H., Lin, C.-S., Lin, L.-Y., 2010. Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae. American Journal of Physiology 298, C237–C250. Zhang, L., Nurse, C.A., Jonz, M.G., Wood, C.M., 2011. Ammonia sensing by neuroepithelial cells and ventilatory responses to ammonia in rainbow trout. Journal of Experimental Biology 214, 2678–2689. Zhang, L., Wood, C.M., 2009. Ammonia as a stimulant to ventilation in rainbow trout Oncorhynchus mykiss. Respiratory Physiology and Neurobiology 168, 261–271.