Flexible ammonia handling strategies using both cutaneous and branchial epithelia in the highly ammonia-tolerant Pacific hagfish.

Hagfish consume carrion, potentially exposing them to hypoxia, hypercapnia, and high environmental ammonia (HEA). We investigated branchial and cutaneous ammonia handling strategies by which Pacific hagfish (Eptatretus stoutii) tolerate and recover from high ammonia loading. Hagfish were exposed to HEA (20 mmol/l) for 48 h to elevate plasma total ammonia (TAmm) levels before placement into divided chambers for a 4-h recovery period in ammonia-free seawater where ammonia excretion (JAmm) was measured independently in the anterior and posterior compartments. Localized HEA exposures were also conducted by subjecting hagfish to HEA in either the anterior or posterior compartments. During recovery, HEA-exposed animals increased JAmm in both compartments, with the posterior compartment comprising ~20% of the total JAmm compared with ~11% in non-HEA-exposed fish. Plasma TAmm increased substantially when whole hagfish and the posterior regions were exposed to HEA. Alternatively, plasma TAmm did not elevate after anterior localized HEA exposure. JAmm was concentration dependent (0.05-5 mmol/l) across excised skin patches at up to eightfold greater rates than in skin sections that were excised from HEA-exposed hagfish. Skin excised from more posterior regions displayed greater JAmm than those from more anterior regions. Immunohistochemistry with hagfish-specific anti-rhesus glycoprotein type c (α-hRhcg; ammonia transporter) antibody was characterized by staining on the basal aspect of hagfish epidermis while Western blotting demonstrated greater expression of Rhcg in more posterior skin sections. We conclude that cutaneous Rhcg proteins are involved in cutaneous ammonia excretion by Pacific hagfish and that this mechanism could be particularly important during feeding.

Ammonia excretion in the Atlantic hagfish (Myxine glutinosa) and responses of an Rhc glycoprotein.

Hagfishes, the most ancient of the extant craniates, demonstrate a high tolerance for a number of unfavorable environmental conditions, including elevated ammonia. Proposed mechanisms of ammonia excretion in aquatic organisms include vesicular NH(4)(+) transport and release by exocytosis in marine crabs, and passive NH(3) diffusion, active NH(4)(+) transport, and paracellular leakage of NH3 or NH(4)(+) across the gills of fishes. Recently, an emerging paradigm suggests that Rhesus glycoproteins play a vital role in ammonia transport in both aquatic invertebrates and vertebrates. This study has identified an Rh glycoprotein ortholog from the gills of Atlantic hagfish. The hagfish Rhcg shares a 56-60% amino acid identity to other vertebrate Rhcg cDNAs. Sequence information was used to produce an anti-hagfish Rhcg (hRhcg) antibody. We have used hRhcg to localize protein expression to epithelial cells of the gill and the skin. In addition, we have quantified hRhcg expression following exposure to elevated plasma ammonia levels. Animals exposed to a 3 mmol/kg NH(4)Cl load resulted in significantly elevated plasma ammonia concentrations compared with controls for up to 4 h postinjection. This correlated with net ammonia excretion rates that were also significantly elevated for up to 4 h postinjection. Rhcg mRNA expression in both the gill and skin was significantly elevated by 15 min and 1 h, respectively, and hRhcg protein expression in gills was significantly elevated at 2, 4, and 8 h postinjection. These results demonstrate a potential role for Rhcg in the excretion of ammonia in the Atlantic hagfish.

Immunohistochemical localization of urea and ammonia transporters in two confamilial fish species, the ureotelic gulf toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus).

This study aims to illustrate potential transport mechanisms behind the divergent approaches to nitrogen excretion seen in the ureotelic toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus). Specifically, we wish to confirm the expression of a urea transporter (UT), which is found in the gill of the toadfish and which is responsible for the unique "pulsing" nature of urea excretion and to localize the transporter within specific gill cells and at specific cellular locations. Additionally, the localization of ammonia transporters (Rhesus glycoproteins; Rhs) within the gill of both the toadfish and midshipman was explored. Toadfish UT (tUT) was found within Na(+)-K(+)-ATPase (NKA)-enriched cells, i.e., ionocytes (probably mitochondria-rich cells), especially along the basolateral membrane and potentially on the apical membrane. In contrast, midshipman UT (pnUT) immunoreactivity did not colocalize with NKA immunoreactivity and was not found along the filaments but instead within the lamellae. The cellular location of Rh proteins was also dissimilar between the two fish species. In toadfish gills, the Rh isoform Rhcg1 was expressed in both NKA-reactive cells and non-reactive cells, whereas Rhbg and Rhcg2 were only expressed in the latter. In contrast, Rhbg, Rhcg1 and Rhcg2 were expressed in both NKA-reactive and non-reactive cells of midshipman gills. In an additional transport epithelium, namely the intestine, the expression of both UTs and Rhs was similar between the two species, with only subtle differences being observed.

Dining on the dead in the deep: Active NH4 + excretion via Na+ /H+ (NH4 + ) exchange in the highly ammonia tolerant Pacific hagfish, Eptatretus stoutii.

AIM: Pacific hagfish are exceptionally tolerant to high environmental ammonia (HEA). Here, we elucidated a cellular mechanism that enables hagfish to actively excrete ammonia against steep ammonia gradients expected to be found inside a decomposing whale carcass. METHODS: Hagfish were exposed to varying concentrations of HEA in the presence or absence of environmental Na+ , while plasma ammonia levels were tracked. 14 C-methylammonium was used as a proxy for NH4 + to measure efflux in whole animals and in isolated gill pouches; the latter allowed us to assess the effects of amiloride specifically on Na+ /H+ exchangers (NHEs) in gill cells. Western blotting and immunohistochemistry were utilized to evaluate the abundance and sub-cellular localization of Rhesus glycoprotein (Rh) channels in the response to HEA. RESULTS: Hagfish actively excreted NH4 + against steep inwardly directed ENH4 + (ΔENH4 + ~ 35 mV) and pNH3 (ΔpNH3 ~ 2000 µtorr) gradients. Active NH4 + excretion and plasma ammonia hypo-regulation were contingent on the presence of environmental Na+ , indicating a Na+ /NH4 + exchange mechanism. Active NH4 + excretion across isolated gill pouches was amiloride-sensitive. Exposure to HEA resulted in decreased abundance of Rh channels in the apical membrane of gill ionocytes. CONCLUSIONS: During HEA exposure, hagfish can actively excrete ammonia against a steep concentration gradient using apical NHEs energized by Na+ -K+ -ATPase in gill ionocytes. Additionally, apical Rh channels are removed from the apical membrane, presumably to reduce ammonia loading from the environment. We suggest that this mechanism allows hagfish to maintain tolerable ammonia levels while feeding inside decomposing carrion, allowing them to exploit nutrient-rich food-falls.

A Field Test of Opportunities for Teen Dating Violence Disclosure in School-Based Relationship Education Programs.

School-based relationship education programs offer an opportunity to identify youth who are experiencing teen dating violence (TDV), support their safety, and connect them with individualized services or referrals. However, no research has tested the feasibility or accuracy of approaches to create opportunities for TDV disclosure in the context of school-based programs. The current study presents the results of a field test comparing three tools used to provide opportunities for TDV disclosure (two questionnaire-style tools and one universal education discussion guide). High school students from two federally funded healthy marriage and relationship education (HMRE) program sites (N = 648) were offered the three tools in random order over the course of the HMRE program, which lasted between 3 weeks and 3 months and took place during the school day. Onsite qualitative interviews with HMRE program staff and their local domestic violence program partners assessed how service providers saw the tools and the process of implementing them. Latent class models examined the accuracy of the tools in identifying TDV. Sensitivities of the tools were low and specificities were high; the questionnaire-style tools tended to have higher sensitivities and fewer classification errors than the universal education tool. Several three-item combinations from across the tools performed better than any intact tool, suggesting that shorter assessments may be effective, provided they include items on sexual coercion and physical violence. Qualitative findings suggested that implementation of TDV assessment and universal education in school settings is a viable strategy, provided programs are able to gain support from school staff, adapt to tight time constraints, and plan procedures for protecting student privacy and confidentiality.

Divergent Pathways of Ammonia and Urea Production and Excretion during the Life Cycle of the Sea Lamprey.

Little is known about nitrogenous waste (N waste) handling and excretion (JN waste) during the complex life cycle of the sea lamprey (Petromyzon marinus), an extant jawless fish that undergoes a complete metamorphosis from a filter-feeding larva (ammocoete) into a parasitic juvenile that feeds on the blood of larger, jawed fishes. Here, we investigate the ammonia- and urea-handling profiles of sea lampreys before, during, and after metamorphosis. The rates of ammonia excretion (Jamm) and urea excretion (Jurea) significantly decreased after the onset of metamorphosis, with the lowest rates observed during midmetamorphosis. Near the completion of metamorphosis, rates of JN waste (JN waste=Jamm+Jurea) significantly increased as sea lampreys entered the juvenile period. Feeding juvenile lampreys had greater than 10- to 15-fold higher Jamm and fivefold higher Jurea compared to nonfed juveniles, which corresponded to higher postprandial (postfeeding) concentrations of plasma ammonia and urea. The routes of Jamm and Jurea completely diverged following metamorphosis. In larvae, Jamm was equally split between branchial (gills) and extrabranchial (skin plus renal) pathways, but following metamorphosis, >80% of ammonia was excreted via the gills in nonfeeding juvenile lampreys, and >95% of ammonia was excreted via the gills in adult sea lampreys. Urea, on the other hand, was predominantly excreted via extrabranchial routes and, to a lesser extent, the gills in larvae and in nonfeeding juveniles. In adults, however, virtually all urea was excreted via urine. Reverse transcription polymerase chain reaction and in silico analyses also indicated that a urea transporter encoded by a slc4a2-like gene is present in lampreys. The branchial expression of this transporter is modulated throughout sea lamprey life history, as it is higher in the larvae and steadily decreases until the adult stage. We conclude that the divergent pathways of Jamm and Jurea during the sea lamprey life cycle reflect changes in their habitat, lifestyle, and diet. Further, the near-complete reliance on renal routes for Jurea in adult sea lampreys is unique among fishes and may reflect the ancestral condition of how this N waste product was handled and excreted by the earliest vertebrates.

Prediction of anxiety symptoms in preschool-aged children: examination of maternal and paternal perspectives.

BACKGROUND: Little is known about risk factors for anxiety in young children. The current study investigated the value of a set of theoretically derived risk factors to predict symptoms of anxiety in a sample of preschool-aged children. METHODS: Mothers (n = 632) and fathers (n = 249) completed questionnaires twice, 12 months apart. Measures were selected to assess several risk factors derived from current theory, including parental negative affectivity, child inhibition, parent overprotection, and impact of life events. RESULTS: Even at this young age anxiety across 12 months was moderately to highly stable (r's = .75 and .74 based on maternal and paternal report respectively). Over and above this stability, according to maternal report, anxiety at 12 months was significantly predicted by prior maternal overprotection, impact of negative life events, child's inhibition, and maternal negative affectivity. According to paternal report, anxiety at 12 months was significantly predicted by prior paternal overprotection and impact of negative life events. The models did not differ significantly for girls and boys. CONCLUSIONS: The results support theories of the development of anxiety, especially the role of parental and external environmental factors, and point to possible targets for prevention of heightened anxiety in young children.

The assessment of anxiety symptoms in preschool-aged children: the revised Preschool Anxiety Scale.

The purpose of this study was to test the validity and factorial structure of a modified version of the Preschool Anxiety Scale (Spence, Rapee, McDonald, & Ingram, 2001). The measure was completed by 764 mothers and 418 fathers of children aged 3 to 5 years. After removing, two items tapping obsessive compulsive symptoms, confirmatory factor analysis showed that a four-factor model (social anxiety, generalized anxiety, separation anxiety, specific fears) all loading on a higher order "anxiety" factor, provided an optimal fit for the data. The total scale and 4 subscales showed strong internal consistency (alphas = .72-.92), 12-month stability y (rs = .60-.75) and maternal/paternal agreement (rs = .60-.75). Scores on the scale also showed expected correlations with a measure of emotional distress, diagnosed anxiety disorders, and behavioral indicators of anxiety.

The curious case of the chemical composition of hagfish tissues--50 years on.

Modern hagfishes are considered to be the most primitive of the living craniates and along with their close jawless agnathan relative, the lamprey, take us back an astonishing 500 million years to the base of the vertebrate evolutionary tree. The unique osmoconforming strategy of the hagfish, whereby the osmotic constituents of the blood plasma bear more of a resemblance to marine invertebrates than vertebrates, has been classically depicted in comparative physiology textbooks for many years. Fifty years ago in this journal, Bellamy and Chester Jones [Bellamy and Chester Jones, 1961. CBP 3, 173-183] published a paper on the chemical composition of the tissues of the Atlantic hagfish, Myxine glutinosa. This publication was one of a flurry of papers published in the 50s, 60s and early 70s focused on describing the ionic and osmotic components of this bizarre fish. Here we take a retrospective look at the research that has taken place on these intriguing animals prior to and following the Bellamy and Chester Jones manuscript, focusing on tissue chemical compositions, the possible role of amino acids, and our current view on ion regulation, metabolism and hypoxia tolerance.

Variation in lethality and effects of two Australian chirodropid jellyfish venoms in fish.

The North Queensland chirodropid box jellyfish Chironex fleckeri and Chiropsalmus sp. share similar nematocyst composition and the same prey of Acetes australis shrimps in their early medusa stages; however, as C. fleckeri individuals reach larger size, the animals add fish to their diet and their complement of nematocyst types changes, allowing larger doses of venom to be delivered to prey. This study demonstrated that the venoms of the two species differ as well: despite similar effects previously documented in crustacean prey models, the two had widely different cardiac and lethal effects in fish, with C. fleckeri being substantially more potent in its ability to cause death. Comparisons between the venom delivery abilities of the two species showed that the change in nematocysts of C. fleckeri cannot alone account for its ontogenetic shift to prey fish; instead, its prey ecology clearly necessitates it having venom capable of acting efficiently to cause death in fish. Although this venom is almost certainly produced at greater metabolic cost to the animal than the less-lethal venom of Chiropsalmus sp., owing to its greater molecular protein complexity, it confers the advantage of increased caloric intake from fish prey, facilitating larger size and potentially greater reproductive output of C. fleckeri over Chiropsalmus sp.

Altering the trajectory of anxiety in at-risk young children.

OBJECTIVE: Increasing evidence for the importance of several risk factors for anxiety disorders is beginning to point to the possibility of prevention. Early interventions targeting known risk for anxiety have rarely been evaluated. The authors evaluated the medium-term (3-year) effects of a parent-focused intervention for anxiety in inhibited preschool-age children. METHOD: The study was a randomized controlled trial of a brief intervention program provided to parents compared with a monitoring-only condition. Participants were 146 inhibited preschool-age children and their parents; data from two or more assessment points were available at 3 years for 121 children. Study inclusion was based on parent-reported screening plus laboratory-observed inhibition. The six-session group-based intervention included parenting skills, cognitive restructuring, and in vivo exposure. The main outcome measures were number and severity of anxiety disorders, anxiety symptoms, and extent of inhibition. RESULTS: Children whose parents received the intervention showed lower frequency and severity of anxiety disorders and lower levels of anxiety symptoms according to maternal, paternal, and child report. Levels of inhibition did not differ significantly based on either parent report or laboratory observation. CONCLUSIONS: This brief, inexpensive intervention shows promise in potentially altering the trajectory of anxiety and related disorders in young inhibited children.

A selective intervention program for inhibited preschool-aged children of parents with an anxiety disorder: effects on current anxiety disorders and temperament.

OBJECTIVE: The current study evaluated the efficacy of early intervention for preschool-aged children selected on the basis of risk who also met diagnostic criteria for anxiety disorders. METHOD: Seventy-one 3- to 4-year-old children were selected based on demonstrating high levels of inhibition and having a parent with a current anxiety disorder. They were randomly allocated to an eight-session parent intervention or waitlist. RESULTS: At baseline, all of the children met criteria for one or more anxiety disorders. At 6-month follow-up, the intervention group showed a significantly greater reduction in anxiety disorders and less interference from their anxiety than the waitlist. In addition, children in the intervention condition showed greater reductions in parent and laboratory observed measures of behavioral inhibition. CONCLUSIONS: The results suggest that a brief early intervention delivered through parents can reduce current anxiety and associated risk and may have the potential to alter the developmental trajectory of anxiety in a high-risk group of young children.

Molecular detection and immunological localization of gill Na+/H+ exchanger in the dogfish (Squalus acanthias).

The dogfish (Squalus acanthias) can make rapid adjustments to gill acid-base transfers to compensate for internal acidosis/alkalosis. Branchial Na+/H+ exchange (NHE) has been postulated as one mechanism driving the excretion of H+ following acidosis. We have cloned gill cDNA that includes an open reading frame coding for a 770-residue protein most homologous (approximately 71%) to mammalian NHE2. RT-PCR revealed NHE2 transcripts predominantly in gill, stomach, rectal gland, intestine, and kidney. In situ hybridization with an antisense probe against NHE2 in gill sections revealed a strong mRNA signal from a subset of interlamellar and lamellae cells. We developed dogfish-specific polyclonal antibodies against NHE2 that detected a approximately 70-kDa protein in Western blots and immunologically recognized branchial cells having two patterns of protein expression. Cytoplasmic and apical NHE2 immunoreactivity were observed in cells coexpressing basolateral Na+-K+-ATPase. Other large ovoid cells more generally staining for NHE2 also were strongly positive for basolateral H+-ATPase. Gill mRNA levels for NHE2 and H+-ATPase did not change following systemic acidosis (as measured by quantitative PCR 2 h after a 1- or 2-meq/kg acid infusion). These data indicate that posttranslational adjustments of NHE2 and other transport systems (e.g., NHE3) following acidosis may be of importance in the short-term pH adjustment and net branchial H+ efflux observed in vivo. NHE2 may play multiple roles in the gills, involved with H+ efflux from acid-secreting cells, basolateral H+ reabsorption for pHi regulation, and in parallel with H+-ATPase for the generation of HCO3(-) in base-secreting cells.

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias.

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.

Na+/H+ antiporter, V-H+-ATPase and Na+/K+-ATPase immunolocalization in a marine teleost (Myoxocephalus octodecemspinosus).

Long-term pH compensation in a marine teleost requires the transepithelial excretion of H(+) across the gill epithelium. H(+) efflux in the longhorn sculpin (Myoxocephalus octodecemspinosus) is dependent on external sodium ion concentration and is inhibited by known inhibitors of Na(+)/H(+) exchangers. Our model for proton transport suggests acid-excreting cells in the gill with an apical Na(+)/H(+) antiporter and basolateral Na(+)/K(+)-ATPase. This model is similar to mammalian kidney and elasmobranch gill epithelium in which a basolateral electrogenic-vacuolar proton pump (V-H(+)-ATPase) localizes to base-excreting cells. The objective of this study was to detect the presence and location of membrane transporters in marine fish gills using immunohistochemical staining. Our data indicate the presence of an apical and subapical Na(+)/H(+)-exchanger 2 (NHE2) in the sculpin gill. NHE2 is present in large, ovoid chloride cells and often colocalizes in the same cells as Na(+)/K(+)-ATPase. We also detected V-H(+)-ATPase immunoreactivity, predominantly in cells at the base of the lamellae, with staining patterns indicative of a basolateral location. The 85 kDa protein detected on immunoblots with anti-NHE2 antibodies was found in both control and acid-infused animals and did not change following a large acute acidosis over 8 h.

Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport.

We have explored the molecular basis for differences in physiological function between the gills and opercular epithelium of the euryhaline killifish Fundulus heteroclitus. These tissues are functionally similar in seawater, but in freshwater the gills actively absorb Na+ but not Cl-, whereas the opercular epithelium actively absorbs Cl- but not Na+. These differences in freshwater physiology are likely due to differences in absolute levels of gene expression (measured using real-time PCR), as several proteins important for Na+ transport, namely Na+,H+-exchanger 2 (NHE2), carbonic anhydrase 2 (CA2), Na+,HCO3- cotransporter 1, and V-type H+-ATPase, were expressed at 3- to over 30-fold higher absolute levels in the gills. In gills, transfer from 10% seawater to freshwater increased the activity of Na+,K+-ATPase by twofold (from 12 h to 7 days), increased the expression of NHE2 (at 12 h) and CA2 (from 12 h to 7 days), and decreased the expression of NHE3 (from 12 h to 3 days). In opercular epithelium, NHE2 was not expressed; furthermore, Na+,K+-ATPase activity was unchanged after transfer to freshwater, CA2 mRNA levels decreased, and NHE3 levels increased. Consistent with their functional similarities in seawater, killifish gills and opercular epithelium expressed Na+,K+-ATPase alpha 1a, Na+,K+,2Cl- cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and the signalling protein 14-3-3a at similar absolute levels. Furthermore, NKCC1 and CFTR were suppressed equally in each tissue after freshwater transfer, and 14-3-3a mRNA increased in both. These results provide insight into the mechanisms of ion transport by killifish gills and opercular epithelia, and demonstrate a potential molecular basis for the differences in physiological function between these two organs.

Stimulation of renal sulfate secretion by metabolic acidosis requires Na+/H+ exchange induction and carbonic anhydrase.

The acute effect of metabolic acidosis on SO(4)(2-) secretion by the marine teleost renal proximal tubule was examined. Metabolic acidosis was mimicked in primary cultures of winter flounder renal proximal tubule epithelium (fPTCs) mounted in Ussing chambers by reducing interstitial pH to 7.1 (normally 7.7). fPTCs with metabolic acidosis secreted SO(4)(2-) at a net rate that was 40% higher than in paired isohydric controls (pH 7.7 on interstitium). The stimulation was completely blocked by the carbonic anhydrase inhibitor methazolamide (100 microM). Although Na(+)/H(+) exchange (NHE) isoforms 1, 2, and 3 were identified in fPTCs by immunoblotting, administering EIPA (20 microM) to the interstitial and luminal bath solutions had no effect on net SO(4)(2-) secretion by fPTCs with a normal interstitial pH of 7.7. However, EIPA (20 microM) blocked most of the stimulation caused by acidosis when applied to the lumen but not interstitium, demonstrating that induction of brush-border NHE activity is important. In the intact flounder, serum pH dropped 0.4 pH units (pH 7.7 to 7.3, at 2-3 h) when environmental pH was lowered from 7.8 to approximately 4.3. Whereas serum [SO(4)(2-)] was not altered by acidosis, renal tubular SO(4)(2-) secretion rate was elevated 200%. Thus metabolic acidosis strongly stimulates renal sulfate excretion most likely by a direct effect on active renal proximal tubule SO(4)(2-) secretion. This stimulation appears to be dependent on inducible brush-border NHE activity.

The effect of environmental hypercapnia and salinity on the expression of NHE-like isoforms in the gills of a euryhaline fish (Fundulus heteroclitus).

The current models for branchial acid excretion in fishes include Na(+)/H(+) exchange and the electrogenic excretion of H+ via H+-ATPase. The predominant route of acid excretion in some freshwater fishes is thought to be via the H+-ATPase/Na+ channel system. The euryhaline Fundulus heteroclitus may not fit this profile even when adapted to freshwater (FW). We hypothesize that the Na+/H+ exchanger (NHE) in this species may play a predominant role in acid-base regulation for both marine and FW adapted animals. Acidosis induced by ambient hypercapnia (1% CO2 in air), resulted in an increase in net H+ excretion to the water in F. heteroclitus pre-adapted to FW, brackish (isoosmotic; BW) and seawater (SW). Both FW and SW adapted mummichogs were tested for NHE protein expression using mammalian NHE antibodies, and we identified NHE-like immunoreactive proteins in gill membrane preparations from both groups. Hypercapnia induced a approximately three-fold elevation in gill NHE2-like protein in FW animals but SW adapted fish showed inconsistent NHE3-like protein expression. There was no change in NHE-1 levels in FW fish. In contrast, SW animals demonstrated a significant increase in both NHE1 and NHE3-like proteins following hypercapnia but limited expression of the NHE2 protein. We hypothesize that different isoforms of NHE may be preferentially expressed depending on the salinity to which the animals are adapted. Net H+ transfers during acidosis may be driven, at least in part by the action of these transporters.

Immunolocalization of Na+/K+-ATPase, carbonic anhydrase II, and vacuolar H+-ATPase in the gills of freshwater adult lampreys, Geotria australis.

As adults, anadromous lampreys migrate from seawater into freshwater rivers, where they require branchial ion (NaCl) absorption for osmoregulation. In teleosts and elasmobranchs, pharmological, immunohistochemical, and molecular data support roles for Na+/K+-ATPase (NPPase), carbonic anhydrase II (CAII), and vacuolar H+-ATPase (V-ATPase) in two different models of branchial ion absorption. To our knowledge, these transport-related proteins have not been studied in adult freshwater lampreys, and therefore it is not known if they are expressed, or have similar functions, in lampreys. The purpose of this study was to localize NPPase, CAII, and V-ATPase in the gills of adult freshwater lampreys and determine if any of these transport-related proteins are expressed in the same cells. Heterologous antibodies were used to localize the three proteins in gill tissue from pouched lamprey (Geotria australis). Immunoreactivity (IR) for all three proteins occurred between, and at the base of, lamellae in cells that match previous descriptions of mitochondrion-rich-cells (MRCs). NPPase-IR was always on the basolateral side of cells that did not stain for CAII or V-ATPase. In contrast, CAII-IR was always on the apical side of cells that also contained diffuse V-ATPase-IR. Therefore, we have identified two types of MRC in adult freshwater lamprey gills based on immunohistochemical staining for three transport proteins. A model of ion transport, based on our results, is proposed for adult freshwater lampreys.

Acid-base regulation in fishes: cellular and molecular mechanisms.

The mechanisms underlying acid-base transfers across the branchial epithelium of fishes have been studied for more than 70 years. These animals are able to compensate for changes to internal pH following a wide range of acid-base challenges, and the gill epithelium is the primary site of acid-base transfers to the water. This paper reviews recent molecular, immunohistochemical, and functional studies that have begun to define the protein transporters involved in the acid-base relevant ion transfers. Both Na(+)/H(+) exchange (NHE) and vacuolar-type H(+)-ATPase transport H(+) from the fish to the environment. While NHEs have been thought to carry out this function mainly in seawater-adapted animals, these proteins have now been localized to mitochondrial-rich cells in the gill epithelium of both fresh and saltwater-adapted fishes. NHEs have been found in the gill epithelium of elasmobranchs, teleosts, and an agnathan. In several species, apical isoforms (NHE2 and NHE3) appear to be up-regulated following acidosis. In freshwater teleosts, H(+)-ATPase drives H(+) excretion and is indirectly coupled to Na(+) uptake (via Na(+) channels). It has been localized to respiratory pavement cells and chloride cells of the gill epithelium. In the marine elasmobranch, both branchial NHE and H(+)-ATPase have been identified, suggesting that a combination of these mechanisms may be utilized by marine elasmobranchs for acid-base regulation. An apically located Cl(-)/HCO(3)(-) anion exchanger in chloride cells may be responsible for base excretion in fresh and seawater-adapted fishes. While only a few species have been examined to date, new molecular approaches applied to a wider range of fishes will continue to improve our understanding of the roles of the various gill membrane transport processes in acid-base balance.