Pacific Hagfish Research Articles

Extrabranchial mechanisms of systemic pH recovery in hagfish (Eptatretus stoutii)

This study investigates the role of branchial and extrabranchial processes in acid–base regulation in the Pacific Hagfish (Eptatretus stoutii). Hagfish were injected with one of the following solutions: acid saline (250mM HCl [pH=0.60], 250mM NaCl), alkaline saline (250mM NaHCO3, 250mM NaCl, [pH≈8.43]) or control saline (500mM NaCl) in order to achieve an acid/alkaline/saline load of 6000μmol·kg−1. Using a custom designed hagfish compartmentalizing flux chamber, we partitioned flux of net acid or base equivalents and ammonia into the anterior (gill+skin) and posterior (skin+intestinal/renal/cloacal) components. We found that Pacific hagfish excrete H+ primarily via branchial mechanisms but base excretion occurs through extrabranchial mechanisms located in the posterior region. In addition, we demonstrate that hagfish are able to excrete ammonia via the skin although this flux was not involved in compensation from an acid–base disturbance.

Phylogeny and effects of anoxia on hyperpolarization-activated, cyclic nucleotide-gated channel gene expression in the heart of a primitive chordate, the Pacific Hagfish (eptatretus stoutii)

The aneural heart of the Pacific hagfish, Eptatretus stoutii, varies heart rate fourfold during recovery from anoxia. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which play an important role in establishing the pacemaker rate of vertebrate hearts, were postulated to be present in this ancestral vertebrate heart, and it was also theorized that changes in hagfish heart rate with oxygen availability involved altered HCN expression. Partial gene cloning revealed six HCN isoforms in the hagfish heart. Hagfish representatives of HCN2, HCN3 and HCN4 were discovered, with HCN2 and HCN3 existing as isoforms designated as HCN2a, HCN2b, HCN3a, two paralogs of HCN3b, and HCN3c. Phylogenetic analysis revealed HCN3b and HCN3c to be ancestral, followed by HCN3a, HCN4 and HCN2. Moreover, HCN3a expression was dominant in both the atrial and ventricular chambers, suggesting that the HCN4 dominance in adult mammalian hearts appeared after hagfish divergence. HCN expression was higher in the atrium than in the ventricle, as might be expected given that atrial beating rate is known to be faster than the ventricular rate. In addition, mRNA expression under normoxic conditions was compared with that following 24 h of anoxia, and either a 2-h or 36-h recovery in normoxic water. In the ventricle, anoxia decreased HCN3a but not HCN4 expression. In contrast, atrial HCN3a expression significantly increased following 2 h of recovery, before returning to control levels following 36 h of recovery, possibly contributing to heart rate changes previously observed under these conditions.

Pharmacological characterization of the heartbeat in an extant vertebrate ancestor, the Pacific hagfish, Eptatretus stoutii

Pharmacological ion-channel blockers were used to investigate the spontaneous heart rates in Pacific hagfish, Eptatretus stoutii. Zatebradine, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, vastly reduced atrial and ventricular contraction rates in a similar concentration-dependent manner, indicating a major role for HCN in setting intrinsic heart rate. When voltage-gated Na+ channels were blocked with tetrodotoxin (TTX), atrial contraction rate declined in a dose-dependent manner, but remained faster than ventricular rate even at very high TTX concentrations. This TTX resistance compared with other fish suggests an important role for a TTX-sensitive inactivation-resistant Na+ current in atrioventricular conduction and chamber synchrony, and a lesser role in setting intrinsic heart rate. T and L-type calcium channel blockers, nickel and nifedipine respectively, also reduced atrial and ventricular contraction rates, nickel having a larger effect on the atrium. These novel results for hagfish are consistent with intrinsic atrial and ventricular rates being set mostly by HCN, with lesser contributions from other ion channels. We suggest that future electrophysiological studies will reveal that hagfishes, with their ancestral position in the evolution of the vertebrate-type chambered heart, share some but not all features of vertebrate intrinsic heart rate control.

Kidney function and sulphate transport in Pacific Hagfish (Eptatretus Stoutii)

The hagfish diverged from vertebrate lineage ~550–600 mya and therefore represent a unique model to study early ionoregulatory strategies. Whilst hagfish are osmoconformers for Na+ and Cl−, these animals have plasma concentrations of the divalent (Ca2+, Mg2+ and S042‐) ions at 1/3 to 1/2 seawater. A difficulty in molecular research involving hagfish is the lack of genomic information. Using a recently obtained hagfish Illumina transcriptome, we have identified a putative sulfate transporter (SLC26a1). We investigated if hagfish possess active S042‐ handling mechanisms by injecting a chronic (3 day) S042‐ load to upregulate potential S042‐ handling and transporting abilities. Using 3H‐ inulin and 35S (as S042‐) injections into the blood, we monitored both GFR and net S042‐ excretion. We also investigated the changes in expression of hfSLC26a1. We determined that Pacific hagfish have a relatively high GFR (~ 0.46 mls/kg/h) despite being osmoconformers and are able to increase sulfate excretion/handling following chronic S042‐loading from 0.06 umol/kg/h to 0.28 umol/kg/h.

Ontogenetic scaling of the morphology and biomechanics of the feeding apparatus in the Pacific hagfish Eptatretus stoutii

The form and function of the support skeleton, musculature and teeth were examined in an ontogenetic series of Pacific hagfish Eptatretus stoutii spanning about a six-fold range in total length (L(T)). Tooth area, feeding apparatus length, basal plate size, theoretical dental plate retractile force, penetration force and applied tooth stress were measured relative to body size. Morphological variables (e.g. tooth area and basal plate size) scaled with positive allometry and functional variables (e.g. retractile force and applied tooth stress) scaled isometrically with L(T). These results suggest that juveniles do not undergo ontogenetic dietary changes and consume functionally equivalent prey to adults, although adults can grasp proportionally larger portions of food. Low tooth stress in juveniles and adults imposes mechanical constraints to puncturing and tearing, which are circumvented by a preference for softer prey tissue or the inclusion of knotting behaviours for reducing tougher prey.

Digestion under Duress: Nutrient Acquisition and Metabolism during Hypoxia in the Pacific Hagfish

Hagfish feed by immersing themselves in the body cavities of decaying animals. This ensures a rich nutrient source for absorption via the gills, skin, and gut, but it may also subject hagfish to reduced levels of dissolved oxygen and elevated levels of the products of biological degradation. This study investigated the impacts of hypoxia and ammonia on the assimilation and metabolism of selected nutrients (glycine, l-alanine, and glucose) in Pacific hagfish (Eptatretus stoutii). Throughout exposure to hypoxia, plasma glucose levels increased. This was not accompanied by an increase in gut glucose transport, which suggests mobilization of glucose from body glycogen stores. Hypoxia preexposure enhanced glycine absorption across the gut and the gill, although l-alanine uptake was unchanged in these tissues. A 24-h period of exposure to hypoxia in hagfish concurrently exposed to waterborne radio-labeled glycine led to a large (5.7-fold) increase in brain glycine accumulation. Preexposure to high levels of waterborne ammonia (10 mM) for 24 h had no impact on gut or skin glycine uptake. These results indicate that hagfish are adapted to maintain nutrient assimilation despite environmental stressors and that tissue-specific absorption of key nutrients such as glycine can even be enhanced in order to sustain critical functions during hypoxia.

Comparative retinal morphology of the platypus

The purpose of this study is to identify evolutionary origin and fate of anatomic features of the duck-billed platypus eye. Eyes from the duck-billed platypus and four key evolutionary basal vertebrates (Pacific hagfish, north hemisphere sea lamprey, and Australian and South American lungfishes) were prepared for light microscopy. In addition to a standard panel of stains, tissues were immunostained against a variety of rod and cone opsins. Finally, published opsin sequences of platypus and several other vertebrate species were aligned and compared with immunohistochemical results. A complete scleral cartilage similar to that seen in birds, reptiles and amphibians encloses the platypus eye. This feature is present in sharks and rays, and in extant relatives of tetrapods, the lungfishes. The choroid lacks a tapetum. The retina is largely avascular and is rod-dominated, with a minority of red- and blue- cone immunoreactive photoreceptors. Like marsupials and many nonmammalian vertebrates, cones contain clear inner segment droplets. Double cones were present, a feature not found in eutherian mammals or marsupials. Evaluation of opsins indicates that red and blue immunoreactive cone opsins, but not rhodopsin, are present in the most basal of the extant species examined, the Pacific hagfish. Rhodopsin appears in the Australian and South American lungfishes, establishing emergence of this pigment in an extant relative of tetrapods. Unlike eyes of eutherian mammals, the platypus eye has retained morphologic features present in early tetrapods such as amphibians and their evolutionarily basal sister group, the lungfishes. These include scleral cartilage, double cones and cone droplets. In the platypus, as in other mammals, rod rhodopsin is the predominant photoreceptor pigment, at expense of the cone system.

Three‐Dimensional Structure of the Nasal Passageway of a Hagfish and its Implications for Olfaction

From high-resolution (65 μm) data acquired by magnetic resonance imaging, we have reconstructed the nasal passageway of a single adult hagfish specimen (probably Eptatretus stoutii). We have used this reconstruction to investigate how the anatomy and morphometry of the nasal passageway influence the olfactory ability of the hagfish. We found that the long, broad section of the passageway preceding the nasal chamber will delay the response to an odor by 1-2 s. Diffusion of odorant to the olfactory epithelium, on which the olfactory sensitivity of an animal depends, will be favored by the relatively large surface area of the olfactory epithelium (∼140 mm(2) ) and a modest expansion in the nasal chamber. Oscillating flow (0.3-0.4 Hz) within the narrow (65-130 μm) sensory channels of the nasal chamber is laminar (Reynolds number ∼ 5) and quasi-steady (Womersley number generally less than one). Distribution of flow over the olfactory epithelium may be aided by: (a) a narrowing before the nasal chamber; (b) partial blockage of the nasal passageway by a protrusion on the central olfactory lamella; and (c) the inward inclination of the olfactory lamellae.

Characterisation of l-alanine and glycine absorption across the gut of an ancient vertebrate

This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of L-alanine and glycine conformed to Michaelis-Menten kinetics. An uptake affinity (K(m); substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J (max)) of 83 nmol cm(-2) h(-1) were described for L-alanine. The K(m) and J(max) for glycine were 2.2 mM and 11.9 nmol cm(-2) h(-1), respectively. Evidence suggested that the pathways of L-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.

Adaptations toin situfeeding: novel nutrient acquisition pathways in an ancient vertebrate

During feeding, hagfish may immerse themselves in the body cavities of decaying carcasses, encountering high levels of dissolved organic nutrients. We hypothesized that this feeding environment might promote nutrient acquisition by the branchial and epidermal epithelia. The potential for Pacific hagfish, Eptatretus stoutii, to absorb amino acids from the environment across the skin and gill was thus investigated. l-alanine and glycine were absorbed via specific transport pathways across both gill and skin surfaces, the first such documentation of direct organic nutrient acquisition in a vertebrate animal. Uptake occurred via distinct mechanisms with respect to concentration dependence, sodium dependence and effects of putative transport inhibitors across each epithelium. Significant differences in the absorbed amino acid distribution between the skin of juveniles and adults were noted. The ability to absorb dissolved organic matter across the skin and gill may be an adaptation to a scavenging lifestyle, allowing hagfish to maximize sporadic opportunities for organic nutrient acquisition. From an evolutionary perspective, hagfish represent a transitory state between the generalized nutrient absorption pathways of aquatic invertebrates and the more specialized digestive systems of aquatic vertebrates.

Metabolism and enzyme activities of hagfish from shallow and deep water of the Pacific Ocean

Although hagfishes are ecologically important members of benthic communities there has been little data available on their metabolism. The oxygen consumption, enzyme activities, and muscle proximate composition of shallow living Eptatretus stoutii and deeper living E. deani were measured to investigate hagfish metabolism. Very low rates of oxygen consumption and both aerobic and anaerobic enzyme activities in the body musculature confirmed the low metabolism of hagfishes. However, significant variation in oxygen consumption existed. E. stoutii had significantly lower rates compared to those of the deeper living E. deani and two other shallow living species for which literature data was used. Both species could regulate their oxygen consumption to very low oxygen concentrations. Epatretus deani, which lives in an oxygen minimum zone, had a significantly lower critical oxygen tension (0.83 kPa) compared to E. stoutii (1.47 kPa). The deeper E. deani had greater lipid stores than E. stoutii which may reflect its deeper habitat and more sporadic food supply.

Anoxic survival of the Pacific hagfish (Eptatretus stoutii)

It is not known how the Pacific hagfish (Eptatretus stoutii) can survive extended periods of anoxia. The present study used two experimental approaches to examine energy use during and following anoxic exposure periods of different durations (6, 24 and 36h). By measuring oxygen consumption prior to anoxic exposure, we detected a circadian rhythm, with hagfish being active during night and showing a minimum routine oxygen consumption (RMR) during the daytime. By measuring the excess post-anoxic oxygen consumption (EPAOC) after 6 and 24h it was possible to mathematically account for RMR being maintained even though heme stores of oxygen would have been depleted by the animal's metabolism during the first hours of anoxia. However, EPAOC after 36h of anoxia could not account for RMR being maintained. Measurements of tissue glycogen disappearance and lactate appearance during anoxia showed that the degree of glycolysis and the timing of its activation varied among tissues. Yet, neither measurement could account for the RMR being maintained during even the 6-h anoxic period. Therefore, two independent analyses of the metabolic responses of hagfish to anoxia exposure suggest that hagfish utilize metabolic rate suppression as part of the strategy for longer-term anoxia survival.

Cardiac responses to anoxia in the Pacific hagfish,Eptatretus stoutii

In the absence of any previous study of the cardiac status of hagfishes during prolonged anoxia and because of their propensity for oxygen-depleted environments, the present study tested the hypothesis that the Pacific hagfish Eptatretus stoutii maintains cardiac performance during prolonged anoxia. Heart rate was halved from the routine value of 10.4±1.3 beats min⁻¹ by the sixth hour of an anoxic period and then remained stable for a further 30 h. Cardiac stroke volume increased from routine (1.3±0.1 ml kg⁻¹) to partially compensate the anoxic bradycardia, such that cardiac output decreased by only 33% from the routine value of 12.3±0.9 ml min⁻¹ kg⁻¹. Cardiac power output decreased by only 25% from the routine value of 0.26±0.02 mW g⁻¹. During recovery from prolonged anoxia, cardiac output and heart rate increased to peak values within 1.5 h. Thus, the Pacific hagfish should be acknowledged as hypoxic tolerant in terms of its ability to maintain around 70% of their normoxic cardiac performance during prolonged anoxia. This is only the second fish species to be so classified.

Ammonia and urea excretion in the Pacific hagfish Eptatretus stoutii: Evidence for the involvement of Rh and UT proteins

The nature of ammonia and urea excretion was examined in the Pacific hagfish ( Eptatretus stoutii), which, under resting conditions, excreted similar quantities of nitrogen as either ammonia or urea. In the presence of high external ammonia (HEA) concentrations, ammonia was taken up at high rates and then excreted at similarly high rates upon return to normal water. However, although elevated by HEA, plasma ammonia levels were maintained at approximately 1–4 μmol N g − 1 , reflecting time-dependent decreases in the rates of ammonia uptake, the possible conversion of ammonia to urea, and the potential active excretion of ammonia against a gradient. Internal injections of NH 4Cl caused marked increases in the rate of ammonia excretion and a delayed increase in urea excretion that may have resulted from increasing urea levels in the plasma. Conversely, when the rate of urea excretion was reduced in the presence of 0.1 mM phloretin, ammonia excretion was significantly elevated. Rates of urea excretion were initially increased by approximately 1000-fold following internal urea injections while the presence of high external urea levels (5–100 mM final concentration) resulted in associated linear increases in plasma urea levels. Using hagfish skin mounted in Ussing chambers, the rate of diffusion of ammonia across the skin exceeded that of urea by approximately four times when equivalent gradients were imposed. Based on western blotting and immunocytochemistry, hagfish gill appears to possess Rh proteins (Rhag, Rhbg and Rhcg1) and urea transporter proteins. Despite the tolerance of hagfish to high levels of ammonia and urea, it is suggested that the presence of ammonia and urea transporter proteins may be required during the period of time hagfish spend in burrows or while feeding, when conditions of high ammonia and/or urea might be encountered.

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na(+) and H(+) to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na(+)/nucleoside and H(+)/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H(+) dependence (all mutants), shift in Na(+):nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na(+)-specific hCNT1, uncoupled Na(+) currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na(+)-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na(+) and/or H(+) electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C-, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11-13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX(3)NEFVA(Y/M/F).

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.

The effects of variable water salinity and ionic composition on the plasma status of the Pacific Hagfish (Eptatretus stoutii)

Hagfish are the most pleisiomorphic extant craniates, and based on the similarity of ionic concentrations between their internal milieu and seawater (SW), they have long been touted as a model for osmo- and ionoconformation. As a result, the lack of direct symmetry between hagfish plasma and the environment with respect to [Na(+)], [Cl(-)], [Mg(2+)], and [Ca(2+)] have been left largely unexplored. In order to determine the capacity of hagfish to regulate their blood compartment, we exposed Pacific hagfish (Eptatretus stoutii) to 24, 32, 40, and 48 g/l salinity for 48 h, as well as to two treatments where a portion of the water [Na(+)] was replaced with either Mg(2+) or Ca(2+) at constant salinity for up to 6 days. Following exposure, we measured plasma ion status, pH, and total carbon dioxide (TCO(2)). As expected, our results indicated that hagfish had no capacity to regulate plasma osmolality, [Na(+)], or [Cl(-)], but they did maintain plasma [Mg(2+)] and [Ca(2+)] nearly constant despite fluctuation of environmental salinity or elevated water [Mg(2+)] and [Ca(2+)] (two- and sevenfold, respectively). Furthermore, exposure to elevated water [Mg(2+)] and [Ca(2+)] resulted in a large increase of plasma TCO(2) with little to no increase of plasma pH. We concluded that hagfish may control plasma [Mg(2+)] and [Ca(2+)] at levels below that of their environment via secretion of HCO(3) (-), similar to the mechanisms described in the intestine of teleosts. We speculate that secretion of HCO(3) (-) likely evolved to maintain plasma [Mg(2+)] and [Ca(2+)] below environmental levels (both of which negatively affect nervous function and muscle contraction if elevated), and was an exaptation for the development of water-absorption mechanisms in the intestine of marine osmoregulators. The ancestors of modern hagfish are thought to have never entered freshwater, thus investigations into their ionoregulatory ability potentially have profound implications regarding the evolution of fishes.

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.

What is the active corticosteroid in primitive aganthan vertebrates? The effects of corticosteroids on gluconeogenesis and magnesium regulation in the Pacific hagfish (Eptatretus stouti)

What is the active corticosteroid in primitive aganthan vertebrates? The effects of corticosteroids on gluconeogenesis and magnesium regulation in the Pacific hagfish (Eptatretus stouti)