Role of cytosolic carbonic anhydrase Ca17a in cardiorespiratory responses to CO2 in developing zebrafish (Danio rerio).

The sensing of environmental fluctuations and initiation of appropriate physiological responses is crucial to homeostasis. Neuroepithelial cells (NECs) in fishes are putative chemoreceptors, resembling mammalian Type I (glomus) cells, that respond in vitro to changes in O2, CO2, NH3, and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO2 sensing owing to its presence in NECs. Zebrafish (Danio rerio) lacking functional Ca17a were generated via CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO2 (hypercapnia). Unfortunately, the homozygous knockout mutants (ca17a-/-) did not survive more than ∼12-14 days postfertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation (fV) and cardiac (fH) frequency in response to hypercapnia (1% CO2) in wild-type (ca17a+/+), heterozygous (ca17a+/-) and ca17a-/- fish were used to investigate Ca17a-dependent CO2 sensing and downstream signaling. Wild-type fish exhibited hyperventilation during hypercapnia as indicated by an increase in fV. In the ca17a-/- fish, the hyperventilatory response was attenuated markedly but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a-/- fish exhibited a significantly lower resting fH that became more pronounced as the fish aged. The decrease in resting fH was prevented ("rescued") when ca17a-/- embryos were injected with ca17a mRNA. Collectively, the results of this study support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

Does blood flow limit acute hypoxia performance in larval zebrafish (Danio rerio)?

Oxygen uptake (MO2) in larval zebrafish prior to maturation of the gill relies on cutaneous O2 transfer. Under normoxic conditions, rates of cutaneous O2 transfer are unaffected by haemoglobin availability but are diminished in fish lacking a functional circulatory system, suggesting that internal convection is critically involved in setting the resting MO2 in zebrafish larvae, even when relying on cutaneous O2 transfer. The reliance of MO2 on blood circulation led to the first objective of the current study, to determine whether loss of internal convection would reduce acute hypoxia performance (as determined by measuring critical PO2; Pcrit) in larval zebrafish under conditions of moderate hypoxia (PO2 = 55 mmHg) at 28.5 and 34 °C. Internal convection was eliminated by preventing development of blood vessels using morpholino knockdown of vascular endothelial growth factor (VEGF); these fish are termed VEGF morphants. Breathing frequency (fV) and heart rate (fH) also were measured (at 28.5 °C) to determine whether any detriment in performance might be linked to cardiorespiratory dysfunction. Although MO2 was reduced in the VEGF morphants, there was no significant effect on Pcrit at 28.5 °C. Raising temperature to 34 °C resulted in the VEGF morphants exhibiting a higher Pcrit than the shams, suggesting an impairment of hypoxia tolerance in the morphants at the higher temperature. The usual robust increase in fV during hypoxia was absent or attenuated in VEGF morphants at 4 and 5 days post fertilization (dpf), respectively. Resting fH was reduced in the VEGF morphants and unlike the sham fish, the morphants did not exhibit hypoxic tachycardia at 4 or 5 dpf. The number of cutaneous neuroepithelial cells (presumptive O2 chemoreceptors) was significantly higher in the VEGF morphants and thus the cardiorespiratory impairment in the morphants during hypoxia was unlikely related to inadequate peripheral O2 sensing.

Expression of ion transport genes in ionocytes isolated from larval zebrafish (Danio rerio) exposed to acidic or Na+-deficient water.

In zebrafish (Danio rerio), a specific ionocyte subtype, the H+-ATPase-rich (HR) cell, is presumed to be a significant site of transepithelial Na+ uptake/acid secretion. During acclimation to environments differing in ionic composition or pH, ionic and acid-base regulations are achieved by adjustments to the activity level of HR cell ion transport proteins. In previous studies, the quantitative assessment of mRNA levels for genes involved in ionic and acid-base regulations relied on measurements using homogenates derived from the whole body (larvae) or the gill (adult). Such studies cannot distinguish whether any differences in gene expression arise from adjustments of ionocyte subtype numbers or transcriptional regulation specifically within individual ionocytes. The goal of the present study was to use fluorescence-activated cell sorting to separate the HR cells from other cellular subpopulations to facilitate the measurement of gene expression of HR cell-specific transporters and enzymes from larvae exposed to low pH (pH 4.0) or low Na+ (5 µM) conditions. The data demonstrate that treatment of larvae with acidic water for 4 days postfertilization caused cell-specific increases in H+-ATPase (atp6v1aa), ca17a, ca15a, nhe3b, and rhcgb mRNA in addition to increases in mRNA linked to cell proliferation. In fish exposed to low Na+, expression of nhe3b and rhcgb was increased owing to HR cell-specific regulation and elevated numbers of HR cells. Thus, the results of this study demonstrate that acclimation to low pH or low Na+ environmental conditions is facilitated by HR cell-specific transcriptional control and by HR cell proliferation.

The role of TASK-2 channels in CO2 sensing in zebrafish (Danio rerio).

Peripheral chemosensitivity in fishes is thought to be mediated by serotonin-enriched neuroepithelial cells (NECs) that are localized to the gills of adults and the integument of larvae. In adult zebrafish (Danio rerio), branchial NECs are presumed to mediate the cardiorespiratory reflexes associated with hypoxia or hypercapnia, whereas in larvae, there is indirect evidence linking cutaneous NECs to hypoxic hyperventilation and hypercapnic tachycardia. No study yet has examined the ventilatory response of larval zebrafish to hypercapnia, and regardless of developmental stage, the signaling pathways involved in CO2 sensing remain unclear. In the mouse, a background potassium channel (TASK-2) contributes to the sensitivity of chemoreceptor cells to CO2. Zebrafish possess two TASK-2 channel paralogs, TASK-2 and TASK-2b, encoded by kcnk5a and kcnk5b, respectively. The present study aimed to determine whether TASK-2 channels are expressed in NECs of larval zebrafish and whether they are involved in CO2 sensing. Using immunohistochemical approaches, TASK-2 protein was observed on the surface of NECs in larvae. Exposure of larvae to hypercapnia caused cardiac and breathing frequencies to increase, and these responses were blunted in fish experiencing TASK-2 and/or TASK-2b knockdown. The results of these experiments suggest that TASK-2 channels are involved in CO2 sensing by NECs and contribute to the initiation of reflex cardiorespiratory responses during exposure of larvae to hypercapnia.

Assessing intracellular pH regulation in H+-ATPase-rich ionocytes in zebrafish larvae using in vivo ratiometric imaging.

The H+-ATPase-rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. Here, a ratiometric imaging technique was developed and validated to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo Gene knockdown or CRISPR/Cas9 knockout approaches were used to evaluate the roles of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (NHE3b; slc9a3.2) and the H+-ATPase (atpv1aa). Additionally, the role of HR cell cytosolic carbonic anhydrase (CAc) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalisation were altered markedly in fish experiencing knockdown/knockout of CAc, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown/knockout experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l-1 NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.

The sensing of respiratory gases in fish: Mechanisms and signalling pathways.

Chemoreception in fish is critical for sensing changes in the chemical composition of the external and internal environments and is often the first step in a cascade of events leading to cardiorespiratory and metabolic adjustments. Of paramount importance is the ability to sense changes in the levels of the three respiratory gases, oxygen (O2), carbon dioxide (CO2) and ammonia (NH3). In this review, we discuss the role of piscine neuroepithelial cells (NEC), putative peripheral chemoreceptors, as tri-modal sensors of O2, CO2 and NH3. Where possible, we elaborate on the signalling pathways linking NEC stimulation to afferent responses, the potential role of neurotransmitters in activating downstream neuronal pathways and the impact of altered levels of the respiratory gases on NEC structure and function. Although serotonin, the major neurotransmitter contained within NECs, is presumed to be the principal agent eliciting the reflex responses to altered levels of the respiratory gases, there is accumulating evidence for the involvement of "gasomitters", a class of gaseous neurotransmitters which includes nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S). Recent data suggest that CO inhibits and H2S stimulates NEC activity whereas NO can either be inhibitory or stimulatory depending on developmental age.

Computational fluid dynamics model of avian tracheal temperature control as a model for extant and extinct animals.

Respiratory evaporative cooling is an important mechanism of temperature control in bird. A computational simulation of the breathing cycle, heat and water loss in anatomical avian trachea/air sac model has not previously been conducted. We report a first attempt to simulate a breathing cycle in a three-dimensional model of avian trachea and air sacs (domestic fowl) using transient computational fluid dynamics. The airflow in the trachea of the model is evoked by changing the volume of the air sacs based on the measured tidal volume and inspiratory/expiratory times for the domestic fowl. We compare flow parameters and heat transfer results with in vivo data and with our previously reported results for a two-dimensional model. The total respiratory heat loss corresponds to about 13-19% of the starvation metabolic rate of domestic fowl. The present study can lend insight into a possible thermoregulatory function in species with long necks and/or a very long trachea, as found in swans and birds of paradise. Assuming the structure of the sauropod dinosaur respiratory system was close to avian, the simulation of the respiratory temperature control (using convective and evaporative cooling) in the extensively experimentally studied domestic fowl may also help in making simulations of respiratory heat control in these extinct animals.

Fish in hot water: hypoxaemia does not trigger catecholamine mobilization during heat shock in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) exposed to an acute heat shock (1 h at 25 °C after raising water temperature from 13 °C to 25 °C over 4 h) mount a significant catecholamine response. The present study investigated the proximate mechanisms underlying catecholamine mobilization. Trout exposed to heat shock in vivo exhibited a significant reduction in arterial O(2) tension, but arterial O(2) concentration was not affected by heat shock, nor was catecholamine release during heat shock prevented by prior and concomitant exposure to hyperoxia (to prevent the fall in arterial O(2) tension). Thus, catecholamine mobilization probably was not triggered by impaired blood O(2) transport. Heat-shocked trout also exhibited an elevation of arterial CO(2) tension coupled with a fall in arterial pH, but these factors are not expected to trigger catecholamine release. The changes in blood O(2) and CO(2) tension occurred despite a significant hyperventilatory response to heat shock. Future studies should investigate whether catecholamine mobilization during heat shock in rainbow trout is triggered by a specific effect of high temperature activating the sympathetic nervous system via a thermosensitive transient receptor potential channel.

Does the presence of a seawater gill morphology induced by dietary salt loading affect Cl(-) uptake and acid-base regulation in freshwater rainbow trout Oncorhynchus mykiss.

The goal of this study was to determine the effect of the changes in gill morphology induced by dietary salt feeding on several aspects of gill function in rainbow trout Oncorhynchus mykiss maintained in fresh water with specific emphasis on Cl(-) uptake (J(IN)Cl(-)) and acid-base regulation. The addition of 11% NaCl to the diet caused J(IN)Cl(-) to be reduced by c. 45% from 214·4 ± 26·7 to 117·3 ± 17·4 µmol kg(-1) h(-1) (mean ± s.e.). Rates of Cl(-) efflux (J(OUT)Cl(-)), net Cl(-) flux (J(NET)Cl(-)), J(NET) Na(+) and plasma levels of Na(+) or Cl(-) were unaffected by salt feeding. On the basis of significant effect of the salt diet on decreasing the maximal uptake rate of Cl(-)(J(MAX)Cl(-)), it would appear that internal salt loading caused a decrease in the number of functional ion transport proteins involved in Cl(-) uptake (e.g. Cl(-) -HCO(3)(-) exchangers) and decreased the transporting capacity of existing proteins. The acid-base regulating capacity of control fish and salt-loaded fish was assessed by monitoring arterial blood acid-base status [partial pressure of CO(2) (PCO(2)), pH and HCO(3)(-)] during exposure to external hypercapnia (nominally 7·5 mm Hg). Both groups of fish exhibited typical compensatory responses to sustained hypercapnia consisting of the gradual accumulation of plasma HCO(3) (-) and thus metabolic restoration within 24 h of the initial respiratory acidosis elicited by hypercapnia. Overall, the results demonstrate that while Cl(-) uptake capacity is reduced in salt-fed fish, there is no associated alteration in acid-base regulating capability.

Compensatory regulation of acid-base balance during salinity transfer in rainbow trout (Oncorhynchus mykiss).

In seawater-acclimated rainbow trout (Oncorhynchus mykiss), base secretion into the intestine is a key component of the intestinal water absorption that offsets osmotic water loss to the marine environment. Acid-base balance is maintained by the matched excretion of acid equivalents via other routes, presumably the gill and/or kidney. The goal of the present study was to examine acid-base balance in rainbow trout upon transfer to more dilute environments, conditions under which base excretion into the intestine is predicted to fall, requiring compensatory adjustments of acid excretion at the gill and/or kidney if acid-base balance is to be maintained. Net acid excretion via the gill/kidney and rectal fluid, and blood acid-base status were monitored in seawater-acclimated rainbow trout maintained in seawater or transferred to iso-osmotic conditions. As predicted, transfer to iso-osmotic conditions significantly reduced base excretion into the rectal fluid (by ~48%). Transfer to iso-osmotic conditions also significantly reduced the excretion of titratable acidity via extra-intestinal routes from 183.4 ± 71.3 to -217.5 ± 42.7 µmol kg(-1) h(-1) (N = 7). At the same time, however, ammonia excretion increased significantly during iso-osmotic transfer (by ~72%) so that the apparent overall reduction in net acid excretion (from 419.7 ± 92.9 to 189.2 ± 76.5 µmol kg(-1 )h(-1); N = 7) was not significant. Trout maintained blood acid-base status during iso-osmotic transfer, although arterial pH was significantly higher in transferred fish than in those maintained in seawater. To explore the mechanisms underlying these adjustments of acid-base regulation, the relative mRNA expression and where possible, activity of a suite of proteins involved in acid-base balance were examined in intestine, gill and kidney. At the kidney, reduced mRNA expression of carbonic anhydrase (CA; cytosolic and membrane-associated CA IV), V-type H(+)-ATPase, and Na(+)/HCO(3) (-) co-transporter were consistent with a reduced role in net acid excretion following iso-osmotic transfer. Changes in relative mRNA expression and/or activity at the intestine and gill were consistent with the roles of these organs in osmotic rather than acid-base regulation. Overall, the data emphasize the coordination of acid-base, osmoregulatory and ionoregulatory processes that occur with salinity transfer in a euryhaline fish.

Roles of cortisol and carbonic anhydrase in acid-base compensation in rainbow trout, Oncorhynchus mykiss.

Fish compensate for acid-base disturbances primarily by modulating the branchial excretion of acid-base equivalents, with a supporting role played by adjustment of urinary acid excretion. The present study used metabolic acid-base disturbances in rainbow trout, Oncorhynchus mykiss, to evaluate the role played by cortisol in stimulating compensatory responses. Trout infused with acid (an iso-osmotic solution of 70 mmol L(-1) HCl), base (140 mmol L(-1) NaHCO(3)) or saline (140 mmol L(-1) NaCl) for 24 h exhibited significant elevation of circulating cortisol concentrations. Acid infusion significantly increased both branchial (by 328 µmol kg(-1) h(-1)) and urinary (by 5.9 µmol kg(-1) h(-1)) net acid excretion, compensatory responses that were eliminated by pre-treatment of trout with the cortisol synthesis inhibitor metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone). The significant decrease in net acid excretion (equivalent to enhanced base excretion) of 203 µmol kg(-1) h(-1) detected in base-infused trout was unaffected by metyrapone treatment. Acid- and base-infusions also were associated with significant changes in the relative mRNA expression of branchial and renal cytosolic carbonic anhydrase (tCAc) and renal membrane-linked CA IV (tCA IV). Cortisol treatment caused changes in CA gene expression that tended to parallel those observed with acid but not base infusion. For example, significant increases in renal relative tCA IV mRNA expression were detected in both acid-infused (~2x) and cortisol-treated (~10x) trout, whereas tCA IV mRNA expression was significantly reduced (~5x) in base-infused fish. Despite changes in CA gene expression in acid- or base-infused fish, neither acid nor base infusion affected CAc protein levels in the gill, but both caused significant increases in branchial CA activity. Cortisol treatment similarly increased branchial CA activity in the absence of an effect on branchial CAc protein expression. Taken together, these findings provide support for the hypothesis that in rainbow trout, cortisol is involved in mediating acid-base compensatory responses to a metabolic acidosis, and that cortisol exerts its effects at least in part through modulation of CA.

Zebrafish (Danio rerio) gill neuroepithelial cells are sensitive chemoreceptors for environmental CO2.

Adult zebrafish exhibit hyperventilatory responses to absolute environmental CO(2) levels as low as 0.13% ( mmHg), more than an order of magnitude lower than the typical arterial levels (40 mmHg) monitored by the mammalian carotid body. The sensory basis underlying the ability of fish to detect and respond to low ambient CO(2) levels is not clear. Here, we show that the neuroepithelial cells (NECs) of the zebrafish gill, known to sense O(2) levels, also respond to low levels of CO(2). An electrophysiological characterization of this response using both current and voltage clamp protocols revealed that for increasing CO(2) levels, a background K(+) channel was inhibited, resulting in a partial pressure-dependent depolarization of the NEC. To elucidate the signalling pathway underlying K(+) channel inhibition, we used immunocytochemistry to show that these NECs express carbonic anhydrase (CA), an enzyme involved in CO(2) sensing in the mammalian carotid body. Further, the NEC response to CO(2) (magnitude of membrane depolarization and time required to achieve maximal response), under conditions of constant pH, was reduced by 50% by the CA-inhibitor acetazolamide. This suggests that the CO(2) detection mechanism involves an intracellular sensor that is responsive to the rate of acidification associated with the hydration of CO(2) and which does not require a change of extracellular pH. Because some cells that were responsive to increasing also responded to hypoxia with membrane depolarization, the present results demonstrate that a subset of the NECs in the zebrafish gill are bimodal sensors of CO(2) and O(2).

Gas transfer in dogfish: a unique model of CO2 excretion.

Carbonic anhydrase (CA) is a zinc metalloenzyme that catalyzes the reversible hydration-dehydration reactions of CO(2). It is present in high abundance in the cytoplasm of vertebrate red blood cells, where it contributes to CO(2) excretion. A membrane-bound CA isoform (CA IV) is also present in the lungs of mammals and reptiles, but plays little role in CO(2) excretion. The gills of teleost fish appear to lack plasma-accessible CA activity. In elasmobranchs, however, evidence gathered using a variety of physiological, biochemical and molecular approaches suggests that CA IV is present in the gills, and that at least in dogfish, this CA IV makes a significant contribution to CO(2) excretion by catalyzing the dehydration of plasma HCO(3)(-). The contribution of CA IV to CO(2) excretion is favoured by unusually high relative plasma buffering that aids in the provision of protons for HCO(3)(-) dehydration. Moreover, reduced emphasis on HCO(3)(-) flux through the red blood cell may reflect the occurrence of a slower turnover cytosolic CA in dogfish. This model of CO(2) excretion, in which HCO(3)(-) dehydration in the red blood cell catalyzed by cytosolic CA and HCO(3)(-) dehydration in the plasma catalyzed by membrane-bound CA IV are of comparable importance, has been described for the dogfish. Further work is required to determine whether it applies to elasmobranch fish as a group.

Carbonic anhydrase expression and CO2 excretion during early development in zebrafish Danio rerio.

Carbonic anhydrase (CA) is critical for CO2 excretion in adult fish, but little is known of the expression or function of CA during early development. The present study examined the hypothesis that, as rates of CO2 production increased during early development in zebrafish (Danio rerio), CA would become necessary for effective CO2 excretion, and that the pattern of CA expression during early development would reflect this transition. Real-time RT-PCR was used to examine the mRNA expression of the two main intracellular CA isoforms over a time course of early development ranging from 0 to 120 h post fertilization (h.p.f.). The mRNA expression of zCAb was generally higher than that of zCAc, particularly during the earliest stages of development. Rates of CO2 excretion increased approximately 15-fold from 24 to 48 h.p.f. whereas rates of O2 uptake increased only 6.7-fold over the same period, indicating a relative stimulation of CO2 excretion over O2 uptake. Treatment of 48 h.p.f. larvae with the CA inhibitor acetazolamide resulted in CO2 excretion rates that were 52% of the value in control larvae, a significant difference that occurred in the absence of any effect on O2 uptake. Antisense morpholino oligonucleotides were used to selectively knock down one or both of the main intracellular CA isoforms. Subsequent measurement of gas transfer rates at 48 h.p.f. indicated that CA knockdown caused a significant relative inhibition of CO2 excretion over O2 uptake, regardless of which cytosolic CA isoform was targeted for knockdown. These results suggest that between 24 h.p.f. and 48 h.p.f., developing zebrafish begin to rely on CA to meet requirements for increased CO2 excretion.

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

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 (JinCl-) preceded the mRNA spike, occurring at 2-5 d.p.f. Raising zebrafish in water with a low [Cl-] caused marked increases in JinCl- 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 JinCl- 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 JinCl- 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.

Effect of histological processing and methacrylate sectioning on the area of gill tissue in teleost.

Deformation of biological tissues may occur during histological processing and results in loss of accuracy when quantitative information about cells, tissues and organs is necessary. In this study, the gill tissue from armored catfish (Pterygoplichthys anisitsi) was quantified in each step of processing using the stereological principles. During processing for glycol methacrylate embedding, gill tissue from shrinks significantly but regains its original dimensions after sectioning.

Evidence that SLC26 anion transporters mediate branchial chloride uptake in adult zebrafish (Danio rerio).

Experiments were performed to test the hypothesis that three members of the SLC26 anion transporter gene family (SLC26a3, A4, and A6; hereafter termed za3, za4, and za6) mediate branchial Cl(-)/HCO(3)(-) exchange in adult zebrafish (Danio rerio). Real-time RT-PCR demonstrated that the gill expressed relatively high levels of za6 mRNA; za3 and za4 mRNA, while present, were less abundant. Also, za4 and za6 were expressed at relatively high levels in the kidney. The results of in situ hybridization or immunocytochemistry (za3 only) experiments performed on gill sections revealed that the SLC26 transporters were predominantly expressed on the filament epithelium (especially within the interlamellar regions) and to a lesser extent on the lamellar epithelium at the base of lamellae. This distribution pattern suggests that the SLC26 anion transporters are localized to mitochondrion-rich cells (ionocytes). Transferring fish to water containing low [Cl(-)] (0.02 mmol/l) resulted in significant increases in branchial SLC26 mRNA expression after 5-10 days of exposure relative to fish raised in normal water [Cl(-)] (0.4 mmol/l); transferring fish to Cl(-)-enriched water (2.0 mmol/l) was without effect on mRNA levels. Transferring fish to water containing elevated levels of NaHCO(3) (10-12.5 mmol/l) caused marked increases in branchial SLC26 mRNA expression between 3 and 10 days of transfer that was associated with a significant 40% increase in Cl(-) uptake (as measured upon return to normal water after 7 days). A decrease in whole body net acid excretion (equivalent to an increase in net base excretion) in fish previously maintained in high [NaHCO(3)] water, concurrent with increases in Cl(-) uptake and SLC26 mRNA levels, suggests a role for these anion transporters in Cl(-) uptake and acid-base regulation owing to their Cl(-)/HCO(3)(-) exchange activities.

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

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 HCO(3)(-) at high rates in the absence of serosal HCO(3)(-)/CO(2), demonstrating that endogenous CO(2) is the principal source of HCO(3)(-) 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 CO(2) hydration to fuel apical anion exchange while membrane-associated, luminal CA activity probably facilitates the conversion of HCO(3)(-) to CO(2). The significance of membrane-bound, luminal CA may be in part to reduce HCO(3)(-) gradients across the apical membrane to further enhance anion exchange and thus Cl(-) absorption and to facilitate the substantial CaCO(3) 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.

Ventilation in Pacific hagfish (Eptatretus stoutii) during exposure to acute hypoxia or hypercapnia.

A technique was developed to measure ventilation in unrestrained Pacific hagfish (Eptatretus stoutii) by inserting and fastening into the nostril a flexible tube fitted with an ultrasonic flow probe. This technique permitted the continuous measurement of ventilation (respiratory) frequency (fR), stroke volume and minute ventilation (.V(E)) in real time in fish exposed to acute hypoxia or hypercapnia. Exposing fish to acute hypoxia (final PW(O2)=21.0 +/- 3.4 mm Hg) caused hypoxaemia and a marked increase in .V(E) of 350+/-71 ml min(-1)kg(-1) (from 235 to 585 ml min(-1)kg(-1)) owing exclusively to an increase in fR of 44+/-7 min(-1) (from 19 to 63 min(-1)). Because O(2) consumption (approximately 0.4 mmol kg(-1)h(-1)) was unaltered during hypoxia, there was an associated marked increase in the ventilation convection requirement from 36.7 to 81.8l mmol(-1). Injecting the O(2) chemoreceptor stimulant NaCN into inspired water (external CN-) or pre-branchial blood (internal CN-) evoked ventilatory responses that were similar to those observed during hypoxia although of a lesser magnitude. With external CN(-), V (E) increased maximally by 146+/-46 ml min(-1)kg(-1) and fR increased by 20+/-2 min(-1). With internal CN-, the maximal increase in .V(E) was 93+/-30 ml min(-1)kg(-1) and fR increased maximally by 19+/-6 min(-1). Exposure to acute hypercapnia (final PwC=7.0+/-0.2 mmHg) caused an increase in V (E) of 169+/-60 ml min(-1)kg(-1). These results provide compelling evidence for chemoreceptor-mediated control of breathing in hagfish and suggest that ventilatory responses to environmental hypoxia and hypercapnia in the vertebrates arose in the myxine lineage.

Membrane-associated carbonic anhydrase in the respiratory system of the Pacific hagfish (Eptatretus stouti).

Like other agnathans, the Pacific hagfish (Eptatretus stouti) lacks red blood cell (RBC) Cl(-)/HCO(3)(-) exchange. Despite this absence of anion exchange, the majority (86.7+/-1.4%) of the total CO(2) carried in the blood is found within the plasma as HCO(3)(-), and thus presumably is inaccessible to RBC carbonic anhydrase (CA). As such, a branchial plasma-accessible CA isozyme in hagfish would be beneficial for mobilizing the considerable plasma HCO(3)(-) stores for CO(2) excretion and blood acid-base balance. The current study used a combination of molecular and biochemical methods to identify two membrane-associated CA isozymes in the respiratory system of E. stouti. Using homology cloning methods, CA IV and XV-like isozymes were identified in the gill and RBC, respectively. Real-time PCR analysis of relative mRNA expression revealed that CA IV was specific to the gill, while CA XV was found in several tissues including the RBC, gill, liver, heart and muscle. Isolation of subcellular fractions of gill and RBC verified the presence of membrane-associated CA activity in each tissue that persisted after standard washing protocols. Unlike CA activity associated with the cytoplasmic fractions, the activity in gill membranes was not inhibited by sodium dodecyl sulphate, while RBC membrane activity was inhibited to a lesser degree than the cytoplasmic fraction. Additionally, incubation of gill membrane fractions with phosphatidylinositol-specific phospholipase C released significant CA activity into the supernatant indicating the presence of a glycophosphatidyl inositol-linkage to the membrane, as found with other CA IV and XV isozymes. These results demonstrate that Pacific hagfish possess gill and RBC plasma-accessible membrane-associated CA that may play important roles in respiratory gas exchange and acid-base balance.