S. F. Perry

Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts

The involvement of the freshwater fish gill chloride cells (CCs) in trans-branchial calcium uptake (JinCa2+) was investigated. This was accomplished by assessing the interspecific relationships between the apical surface area of CCs exposed to the external environment and JinCa2+. Three species of freshwater teleosts, the rainbow trout (Oncorhynchus mykiss), the American eel (Anguilla rostrata) and the brown bullhead catfish (Ictalurus nebulosus), were used. Chronic (ten-day) treatment with cortisol in each species was used as a tool to evoke variations in both JinCa2+ and gill CC morphology in order to assess intraspecific relationships between CC surface area and JinCa2+. The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that catfish possessed the lowest fractional area of exposed CC (CCFA) on the gill filament epithelium (12,744 ± 2248 μm2/mm2) and was followed, in increasing order, by American eel (21,355 ± 981 μm2/mm2) and rainbow trout (149,928 ± 26,545 μm2/mm2). With the exception of catfish, chronic treatment with cortisol caused significant increases in CCFA owing to proliferation of CCs and/or enlargement of individual CCs (eel only). The rates of JinCa2+ closely reflected the CC fractional area in each species. The results of correlation analysis revealed significant correlations between CC fractional area and JinCa2+ in trout and eel. Owing to the absence of an effect of cortisol treatment, there was no significant correlation in catfish because of insufficient variation in CC fractional area in this species. CC fractional area was significantly correlated with JinCa2+ among the three species examined. These results suggest that CC is involved in calcium uptake in freshwater teleosts and that both intra- and interspecific differences in the rates of calcium uptake can be accounted for by variability in the surface area of exposed CCs on the gill epithelia.

Chemoreceptor plasticity and respiratory acclimation in the zebrafish Danio rerio.

SUMMARY The goals of this study were to assess the respiratory consequences of exposing adult zebrafish Danio rerio to chronic changes in water gas composition (hypoxia, hyperoxia or hypercapnia) and to determine if any ensuing effects could be related to morphological changes in branchial chemoreceptors. To accomplish these goals, we first modified and validated an established non-invasive technique for continuous monitoring of breathing frequency and relative breathing amplitude in adult fish. Under normal conditions 20% of zebrafish exhibited an episodic breathing pattern that was composed of breathing and non-breathing (pausing/apneic) periods. The pausing frequency was reduced by acute hypoxia (PwO2<130 mmHg) and increased by acute hyperoxia (PwO2>300 mmHg), but was unaltered by acute hypercapnia. Fish were exposed for 28 days to hyperoxia (PwO2>350 mmHg), or hypoxia (PwO2=30 mmHg) or hypercapnia (PwCO2=9 mmHg). Their responses to acute hypoxia or hypercapnia were then compared to the response of control fish kept for 28 days in normoxic and normocapnic water. In control fish, the ventilatory response to acute hypoxia consisted of an increase in breathing frequency while the response to acute hypercapnia was an increase in relative breathing amplitude. The stimulus promoting the hyperventilation during hypercapnia was increased PwCO2 rather than decreased pH. Exposure to prolonged hyperoxia decreased the capacity of fish to increase breathing frequency during hypoxia and prevented the usual increase in breathing amplitude during acute hypercapnia. In fish previously exposed to hyperoxia, episodic breathing continued during acute hypoxia until PwO2 had fallen below 70 mmHg. In fish chronically exposed to hypoxia, resting breathing frequency was significantly reduced (from 191±12 to 165±16 min–1); however, the ventilatory responses to hypoxia and hypercapnia were unaffected. Long-term exposure of fish to hypercapnic water did not markedly modify the breathing response to acute hypoxia and modestly blunted the response to hypercapnia. To determine whether branchial chemoreceptors were being influenced by long-term acclimation, all four groups of fish were acutely exposed to increasing doses of the O2 chemoreceptor stimulant, sodium cyanide, dissolved in inspired water. Consistent with the blunting of the ventilatory response to hypoxia, the fish pre-exposed to hyperoxia also exhibited a blunted response to NaCN. Pre-exposure to hypoxia was without effect whereas prior exposure to hypercapnia increased the ventilatory responses to cyanide. To assess the impact of acclimation to varying gas levels on branchial O2 chemoreceptors, the numbers of neuroepithelial cells (NECs) of the gill filament were quantified using confocal immunofluorescence microscopy. Consistent with the blunting of reflex ventilatory responses, fish exposed to chronic hyperoxia exhibited a significant decrease in the density of NECs from 36.8±2.8 to 22.7±2.3 filament–1.

Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss).

SUMMARY The entry of calcium (Ca2+) through an apical membrane epithelial calcium channel (ECaC) is thought to a key step in piscine branchial Ca2+ uptake. In mammals, ECaC is a member of the transient receptor potential (TRP) gene family of which two sub-families have been identified, TRPV5 and TPRV6. In the present study we have identified a single rainbow trout (Oncorhynchus mykiss) ECaC (rtECaC) that is similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis of the protein sequence suggests that an ancestral form of the mammalian genes diverged from those in the lower vertebrates prior to the gene duplication event that gave rise to TRPV5 and TRPV6. The putative model for Ca2+ uptake in fish proposes that the mitochondria-rich cell (also termed ionocyte or chloride cell) is the predominant or exclusive site of transcellular Ca2+ movements owing to preferential localisation of ECaC to the apical membrane of these cells. However, the results of real-time PCR performed on enriched gill cell populations as well as immunocytochemistry and in situ hybridisation analysis of enriched cells, cell cultures and whole gill sections strongly suggest that ECaC is not exclusive to mitochondria-rich cells but that it is also found in pavement cells. Not only was ECaC protein localized to areas of the gill normally having few mitochondria-rich cells, but there was also no consistent co-localization of ECaC- and Na+/K+-ATPase-positive (a marker of mitochondria rich cells) cells. Taken together, the results of the present study suggest that although ECaC (mRNA and protein) does exist in trout gill, its cellular distribution is more extensive than previously thought, thus suggesting that Ca2+ uptake may not be restricted to mitochondria-rich cells as was proposed in previous models.

The involvement of H+-ATPase and carbonic anhydrase in intestinal HCO3- secretion in seawater-acclimated rainbow trout.

SUMMARY Pyloric caeca and anterior intestine epithelia from seawater-acclimated rainbow trout exhibit different electrophysiological parameters with lower transepithelial potential and higher epithelial conductance in the pyloric caeca than the anterior intestine. Both pyloric caeca and the anterior intestine secrete HCO3– at high rates in the absence of serosal HCO3–/CO2, demonstrating that endogenous CO2 is the principal source of HCO3– under resting control conditions. Apical, bafilomycin-sensitive, H+ extrusion occurs in the anterior intestine and probably acts to control luminal osmotic pressure while enhancing apical anion exchange; both processes with implications for water absorption. Cytosolic carbonic anhydrase (CAc) activity facilitates CO2 hydration to fuel apical anion exchange while membrane-associated, luminal CA activity probably facilitates the conversion of HCO3– to CO2. The significance of membrane-bound, luminal CA may be in part to reduce HCO3– gradients across the apical membrane to further enhance anion exchange and thus Cl– absorption and to facilitate the substantial CaCO3 precipitation occurring in the lumen of marine teleosts. In this way, membrane-bound, luminal CA thus promotes the absorption of osmolytes and reduction on luminal osmotic pressure, both of which will serve to enhance osmotic gradients to promote intestinal water absorption.

Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss: comparative physiology and molecular evolution.

SUMMARY It is well established that the gills of teleost fish contain substantial levels of cytoplasmic carbonic anhydrase (CA), but it is unclear which CA isozyme(s) might be responsible for this activity. The objective of the current study was to determine if branchial CA activity in rainbow trout was the result of a general cytoplasmic CA isozyme, with kinetic properties, tissue distribution and physiological functions distinct from those of the red blood cell (rbc)-specific CA isozyme. Isolation and sequencing of a second trout cytoplasmic CA yielded a 780 bp coding region that was 76% identical with the trout rbc CA (TCAb), although the active sites differed by only 1 amino acid. Interestingly, phylogenetic analyses did not group these two isozymes closely together, suggesting that more fish species may have multiple cytoplasmic CA isozymes. In contrast to TCAb, the second cytoplasmic CA isozyme had a wide tissue distribution with high expression in the gills and brain, and lower expression in many tissues, including the red blood cells. Thus, unlike TCAb, the second isozyme lacks tissue specificity and may be expressed in the cytoplasm of all cells. For this reason, it is referred to hereafter as TCAc (trout cytoplasmic CA). The inhibitor properties of both cytoplasmic isozymes were similar (Ki acetazolamide 1.21±0.18 nmol l-1 and 1.34±0.10 nmol l-1 for TCAc and TCAb, respectively). However, the turnover of TCAb was over three times greater than that of TCAc (30.3±5.83 vs 8.90±1.95 e4 s-1, respectively), indicating that the rbc-specific CA isoform was significantly faster than the general cytoplasmic isoform. Induction of anaemia revealed differential expression of the two isozymes in the red blood cell; whereas TCAc mRNA expression was unaffected, TCAb mRNA expression was significantly increased by 30- to 60-fold in anaemic trout.

Proton pumps in the fish gill and kidney

The proton pump or vacuolar type H+-ATPase is an oligomeric protein responsible for electrogenic H+ secretion in a variety of acid-secreting epithelia. Recently, the proton pump was identified in both the gill and kidney of freshwater-adapted rainbow trout (Oncorhynchus mykiss). Using immunocytochemistry, H+-ATPase has been localized in the pavement cells and chloride cells of the lamellar epithelium. During periods of internal acidosis, there is a marked increase in the expression of the branchial proton pump as identified by Western analysis, immunocytochemistry and in situ hybridization. This augmented expression of proton pumps occurs concomitantly with a marked increase in branchial acid excretion and Na+ uptake. Immunocytochemical studies suggest that the pavement cell, rather than the chloride cell, is the predominant site of acid excretion during periods of acidosis. These findings are consistent with the notion that in freshwater teleosts, Na+ uptake and H+ excretion are linked via the coupling of the electrogenic proton pump to apical membrane Na+ channels. This mechanism may be controlled by hormones including cortisol and/or growth hormone. The fish kidney plays an important role in regulating acidosis via the re-absorption of filtered HCO3-. Recently, we have demonstrated using Western analysis and immunocytochemistry, the presence of proton pump in rainbow trout kidney and observed increased H+-ATPase expression during respiratory acidosis. These new findings suggest a role for the renal proton pump in acid-base regulation.

The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio) larvae.

SUMMARY After demonstrating phylogenetic relatedness to orthologous mammalian genes, tools were developed to investigate the roles of three members (A3, A4 and A6c) of the SLC26 anion exchange gene family in Cl– uptake and HCO3 excretion in embryos and larvae of zebrafish (Danio rerio). Whole-mount in situ hybridization revealed the presence of SLC26 mRNA in gill primordia, mesonephros and heart (slc26a3 and a4 only) at 5–9 days postfertilization (d.p.f.). SLC26A3 protein was highly expressed in lateral line neuromasts and within the gill, was localized to a sub-population of epithelial cells, which often (but not always) coexpressed Na+/K+-ATPase. SLC26 mRNA levels increased with developmental age, peaking at 5–10 d.p.f.; the largest increases in rates of Cl– uptake (batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (J_{mathrm{in}}^{mathrm{Cl}^{-}}) end{document}) preceded the mRNA spike, occurring at 2–5 d.p.f. Raising zebrafish in water with a low [Cl–] caused marked increases in batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (J_{mathrm{in}}^{mathrm{Cl}^{-}}) end{document} at 3–10 d.p.f. and was associated with increased levels of SLC26 mRNA. Raising fish in water of high [Cl–] was without effect on batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (J_{mathrm{in}}^{mathrm{Cl}^{-}}) end{document} or SLC26 transcript abundance. Selective gene knockdown using morpholino antisense oligonucleotides demonstrated a significant role for SLC26A3 in Cl– uptake in larval fish raised in control water and roles for A3, A4 and A6c in fish raised in water with low [Cl–]. Prolonged (7 days) or acute (24 h) exposure of fish to elevated (2 or 5 mmol l–1) ambient [HCO3–] caused marked increases in Cl– uptake when determined in water of normal [HCO3–] that were accompanied by elevated levels of SLC26 mRNA. The increases in batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (J_{mathrm{in}}^{mathrm{Cl}^{-}}) end{document} associated with high ambient [HCO3–] were not observed in the SLC26 morphants (significant only at 5 mmol l–1 HCO3– for A4 and 2 mmol l–1 HCO3– for A6c). Net base excretion was markedly inhibited in the slc26a3 and a6c morphants thereby implicating these genes in Cl–/HCO3– exchange. The results suggest that under normal conditions, Cl– uptake in zebrafish larvae is mediated by SLC26A3 Cl–/HCO3– exchangers but under conditions necessitating higher rates of high affinity Cl– uptake, SlC26A4 and SLC26A6c may assume a greater role.

Modulation of catecholamine storage and release by the pituitary-interrenal axis in the rainbow trout, Oncorhynchus mykiss

This study examined the effects of pituitary-interrenal hormones on catecholamine storage and release in the rainbow trout Oncorhynchus mykiss. An extract of trout pituitary elicited the release of adrenaline, but not noradrenaline, using an in situ perfusion preparation. A variety of doses of adrenocorticotropic hormone (2–2000 mU) caused the release of both catecholamines in situ which was unaffected by pre-treatment with the ganglion blocker, hexamethonium, or the serotonergic receptor antagonist, methysergide, but was abolished in calcium-free media. Intra-arterial injections of adrenocorticotrophic hormone in vivo caused an elevation of plasma adrenaline but not noradrenaline levels. Injections of cortisol in situ did not elicit catecholamine release. Trout given an intraperitoneal implant of cortisol (50 mg·kg-1 body weight) had significantly higher plasma cortisol concentrations when compared to controls after 7 days of implantation. Increases in the levels of stored catecholamines were observed in various regions of the kidney and posterior cardinal vein following 3 and 7 days of cortisol treatment. The ability of the chromaffin cells to release catecholamines in response to cholinergic stimulation was assessed in situ after 7 days of treatment. Basal (non-stimulated) adrenaline outflowing perfusate levels were greater in the cortisol-treated fish. Cortisol treatment increased the responsiveness of the catecholamine release process to low doses of the cholinoceptor agonist carbachol. Three or 7 days of cortisol treatment did not alter the in vitro activity of the enzyme phenylethanolamine-N-methyl-transferase. The results of this study demonstrate that interactions within the pituitary-adrenal axis can influence both catecholamine storage and release in the rainbow trout.

Control of breathing in African lungfish (Protopterus dolloi): A comparison of aquatic and cocooned (terrestrialized) animals

African lungfish, Protopterus dolloi exhibited constant rates of O(2) consumption before (0.95+/-0.07 mmol kg(-1) h(-1)), during (1.21+/-0.32 mmol kg(-1) h(-1)) and after (1.14+/-0.14 mmol kg(-1) h(-1)) extended periods (1-2 months) of terrestrialization while cocooned. Although a breathing event in terrestrialized fish consisted of multiple bouts of inspiration and expiration in rapid succession, the mean frequency of pulmonary breathing events was unaltered in the terrestrialized fish (16.7+/-1.4 h(-1)versus 20.1+/-4.9 h(-1) in the aquatic and terrestrialized fish, respectively). Hypoxia (approximately 20 mmHg) increased the frequency of breathing events by 16 and 23 h(-1) in the aquatic and terrestrialized fish, respectively. Hyperoxia (approximately 550 mmHg) decreased breathing event frequency by 10 and 15 h(-1) in the aquatic and terrestrialized animals. Aquatic hypercapnia (approximately 37.5 mmHg) increased pulmonary breathing frequency (from 15.3+/-2.3 to 28.7+/-5.4 h(-1)) in free swimming lungfish, whereas aerial hypercapnia was without effect in aquatic or terrestrialized fish.

Developmental plasticity of ventilatory control in zebrafish, Danio rerio.

To determine whether development of ventilatory control in zebrafish (Danio rerio) exhibits plasticity, embryos were exposed to hypoxia, hyperoxia or hypercapnia for the first 7 days post-fertilization. Their acute reflex breathing responses to ventilatory stimuli (hypoxia, hypercapnia and external cyanide) were assessed when they had reached maturity (3 months or older). Zebrafish reared under hyperoxic conditions exhibited significantly higher breathing frequencies at rest (283+/-27min(-1) versus 212+/-16min(-1) in control fish); breathing frequency was unaffected in adult fish subjected to hyperoxia for 7 days. The respiratory responses of fish reared in hyperoxic water to acute hypoxia, hypercapnia or external cyanide were blunted (hypoxia, cyanide) or eliminated (hypercapnia). Adult fish exposed for 7 days to hyperoxia showed no change in acute responses to these stimuli. The respiratory responses to acute hypoxia, hypercapnia or external cyanide of fish reared under hypoxic or hypercapnic conditions were similar to those in fish reared under normal conditions. A subset of all fish examined exhibited episodic breathing; an analysis of breathing patterns demonstrated that fish reared under hypercapnic conditions had an increased tendency to display episodic breathing. The results of this study reveal that there is flexibility in the design and functioning of the embryonic or larval respiratory system in zebrafish.