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 µmolNg⁻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 NH4Cl 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.

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.

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.

Changes in plasma catecholamine concentration during salinity manipulation and anaesthesia in the hagfish Eptatretus cirrhatus.

Plasma catecholamines were measured following surgery under anaesthesia and after exposing hagfish to 90 and 110% sea water (SW). Plasma noradrenaline (NA) concentration increased from a resting value of 7 to 818 nM l(-1) on anaesthesia. Plasma adrenaline (AD) did not change. NA concentrations also increased during volume depletion (110% SW), but to much lower values (26 nM l(-1 )at 100 min). AD concentrations were increased at 20 min, then fell. During volume loading (90% SW) NA fell, and AD increased to a maximum concentration of 511 nM l(-1) at 40 min (resting concentration 24 nM l(-1)). The data are consistent with a vasoconstrictory role for NA on central veins when venous pressures fall and a vasodilatory role for AD on volume expansion.

Molecular cloning and structural characterization of the hagfish proteinase inhibitor of the alpha-2-macroglobulin family.

The "most primitive" living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human alpha2-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg833) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg833 was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.

Water regulates oxygen binding in hagfish (Myxine glutinosa) hemoglobin.

Hagfish hemoglobin (Hb) is considered to represent a transition stage between invertebrate and vertebrate hemoglobins. The Hb system of Myxine glutinosa consists of three monomeric hemoglobins, which upon deoxygenation associate to form primarily heterodimers and heterotetramers. Myxine glutinosa is an osmoconformer, whose red blood cells show the exceptional ability to swell and remain swollen under hyposmotic conditions. In order to determine whether water activity regulates hemoglobin function, the effect of changes in osmolality on hemoglobin-O(2) affinity was investigated by applying the osmotic stress method to purified hemoglobins as well as intact red blood cells. Oxygen affinity decreases when water activity increases, indicating that water molecules stabilize the low-affinity, oligomeric state of the hemoglobin. This effect is opposite to that observed in tetrameric vertebrate hemoglobins, but resembles that seen in the dimeric hemoglobin of the marine clam Scapharca inaequivalvis. Our data show that water may act as an allosteric effector for hemoglobin within intact red cells and even in animals that do not experience large variations in blood osmolality.

Comparative analysis of autoxidation of haemoglobin.

Autoxidation of oxyhaemoglobin (oxyHb) to methaemoglobin was measured at different temperatures in haemoglobin solutions from Atlantic hagfish, river lamprey, common carp, yellowfin tuna and pig. The aims were to evaluate the impact of the absent distal histidine in hagfish haemoglobin, the importance of oxyHb being either monomeric (hagfish and lamprey) or tetrameric (carp, tuna and pig) and to gain information on the temperature-sensitivity of autoxidation. The rate of autoxidation was lower in hagfish than in carp, yellowfin tuna and lamprey haemoglobins at any given temperature. Substitution of the distal histidine residue (His E7) with glutamine in hagfish haemoglobin was therefore not associated with an accelerated autoxidation, as might be expected on the basis of the normal protective role of His E7. Glutamine may have similar qualities to histidine and be involved in the low susceptibility to autoxidation. The low oxidation rate of hagfish haemoglobin, together with an oxidation rate of lamprey haemoglobin that did not differ from that of carp and yellowfin tuna haemoglobins, also revealed that autoxidation was not accelerated in the monomeric oxyhaemoglobins. Pig haemoglobin was oxidised more slowly than fish haemoglobins, demonstrating that fish haemoglobins are more sensitive to autoxidation than mammalian haemoglobins. The rate of autoxidation of hagfish haemoglobin was, however, only significantly greater than that of pig haemoglobin at high temperatures. Autoxidation was accelerated by rising temperature in all haemoglobins. Arrhenius plots of carp and yellowfin tuna haemoglobin revealed a break at 25 degrees C, reflecting a lower temperature-sensitivity between 5 and 25 degrees C than between 25 and 40 degrees C.

Hagfish (Myxine glutinosa) red cell membrane exhibits no bicarbonate permeability as detected by (18)O exchange.

The bicarbonate permeability of the plasma membrane of intact hagfish (Myxine glutinosa) red blood cells and the intracellular carbonic anhydrase activity of these cells were determined by applying the (18)O exchange reaction using a special mass spectrometric technique. When the macromolecular carbonic anhydrase inhibitor Prontosil-Dextran was used to suppress any extracellular carbonic anhydrase activity, the mean intracellular acceleration of the CO(2) hydration/HCO(3)(-) dehydration reaction over the uncatalyzed reaction (referred to as intracellular carbonic anhydrase activity A(i)) was 21 320+/-3000 at 10 degrees C (mean +/- s.d., N=9). The mean bicarbonate permeability of the red blood cell membrane (P(HCO3)-) was indistinguishable from zero. It can be concluded that CO(2) transport within hagfish blood does not follow the classical scheme of CO(2) transport in vertebrate blood. It is suggested that the combination of considerable intraerythrocytic carbonic anhydrase activity and low P(HCO3)- may serve to enhance O(2) delivery to the tissue in the exceptionally hypoxia-tolerant hagfish.

Calcitonin-like substance in plasma of the hagfish, Eptatretus burgeri (Cyclostomata).

Cyclostomes have been regarded as having no ultimobranchial gland. However, cells producing immunoreactive calcitonin (CT) were recently found in cyclostome brains. In the present study, we examined using biochemical and biological methods whether there is a CT-like substance in the plasma of the hagfish, Eptatretus burgeri. Hagfish plasma was first subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) and then separated into twenty fractions. The presence of immunoreactive CT in each fraction was then investigated by Western blotting with salmon CT antiserum. Two fractions (36-39% CH3CN; 39-42% CH3CN) showed positive immunoreactivity. Hypocalcemic and hypophosphatemic activities were detected by a rat bioassay only in the former fraction (36-39% CH3CN). The molecular weight (MW) of the CT-like substance in this fraction was 3.5 kDa, which is equal to that of genuine CT. Furthermore, when hagfish plasma was examined by enzyme-linked immunosorbent assay using anti-salmon CT, the dilution curve of the plasma paralleled the standard curve of salmon CT. The CT-like substance detected here was present at a high concentration (14 ng/ml) in the plasma. From the results of the present study, the CT-like substance present in hagfish plasma appears to be very similar to salmon CT.

Insulin binding and internalization in hagfish red blood cells.

Binding of porcine 125I-insulin (0.15 nM) to hagfish red blood cells was time-dependent, reaching equilibrium after 1 hr at 10 degrees. The specific 125I-insulin binding to hagfish red blood cells was reversible, and unlabeled insulin accelerated the dissociation of 125I-insulin bound to receptors from a T1/2 of 60 min in cells suspended in medium alone to 23 min in medium containing 8 microM nonradioactive insulin. Porcine insulin and desoctapeptide insulin competed for specific binding of 125I-insulin in a dose-dependent manner, whereas glucagon and somatostatin did not. For porcine insulin, Scatchard analysis produced a curvilinear plot, suggesting multiple affinity binding sites with high-affinity and low-affinity association constants (Ka) 0.2 x 10(9) M-1 and 0.27 x 10(7) M-1, respectively. A total of 2090 binding sites per hagfish red blood cell was calculated. Sixty-two percent of the bound 125I-insulin was found to be internalized into the hagfish red blood cells. Less degradation of 125I-insulin was observed by Sephadex G-50 chromatography compared to human red blood cells.

The hemoglobin system of the hagfish Myxine glutinosa: aggregation state and functional properties.

Hemoglobin (Hb) from the hagfish Myxine glutinosa is composed of six major monomeric subunits. Some of these subunits aggregate to dimers at low pH, and to tetramers when deoxygenated and at high protein concentration. The aggregation is inhibited by the presence of KCl. Oxygen equilibrium studies show the presence of a small Bohr effect which is strongly reduced by KCl, indicating that it originates from pH-dependent aggregation. ATP and DPG cause a similar decrease in the Bohr effect. O2 affinity is dependent on protein concentration, temperature and presence of CO2. Cooperativity is practically absent. O2 binding properties of the separated aggregating and non-aggregating Hbs purified at low pH cannot account for the functional properties of the composite hemolysate, suggesting the presence of other subunits interactions. The results are discussed in relation to literature data for other cyclostome Hbs and for M. glutinosa Hb, where the presence of three major monomeric Hbs and a possible CO2-dependent aggregation had been reported.

A cell-surface opsonic receptor on leucocytes from the phylogenetically primitive vertebrate, Eptatretus stouti.

A humoral recognition molecule that is homologous to the mammalian complement components C3, C4 and C5 has recently been identified in the Pacific hagfish, Eptatretus stouti. One function of this complement-like protein (CLP) is to opsonize foreign material for phagocytosis by hagfish leucocytes. Here, we demonstrate that CLP's opsonic activity can be abrogated by pre-incubating phagocytes with an anti-hagfish leucocyte mAb (1B1). Moreover, antigen-activated CLP can block the binding of the 1B1 antibody to hagfish leucocytes. Flow cytometry and immunoprecipitation indicate that 1B1 recognizes a 105 kDa cell-surface, monomeric protein that is expressed exclusively on phagocytic hagfish leucocytes. It is concluded that this 105 kDa protein represents the cell surface receptor by which CLP mediates the phagocytosis of opsonized targets.

Chemotactic responses of hagfish (Vertebrata, Agnatha) leucocytes.

The chemotactic responses of hagfish leucocytes were tested using a variety of chemoattractants. Leucocyte migration was significantly enhanced by purified mammalian complement anaphylotoxin (C5a) and LPS-activated hagfish plasma. Checkerboard analyses confirmed that the responses of leucocytes to both of these chemoattractants were directed along concentration gradients (chemotaxis) and did not result from accelerated random movement (chemokinesis). Chemotaxis was undertaken by leucocyte fractions that were enriched in granulocytes, the predominant phagocytic cells of hagfish. The data suggest that chemotactic mechanisms may have been conserved during evolution to such a degree that mammalian chemoattractants can bind and activate chemotactic receptors on hagfish leucocytes. Moreover, hagfish appear to express plasma proteins that are structurally and functionally homologous to mammalian complement anaphylotoxins.

Cardiovascular responses to hypoxia in the hagfish, Eptatretus cirrhatus.

Simultaneous measurements of cardiac output (Q), blood pressures and blood gases were made in the hagfish, Eptatretus cirrhatus, during exposure to hypoxia. The partial pressure of oxygen in the medium (PIO2) was reduced from 20.7 kPa to 8.0 kPa and then lowered to 5.3 kPa. At a PIO2 of 5.3 kPa there was a 40% increase in Q. Part of the increase may have been due to the increased activity of the animal at low PIO2. In recovery, when the animals were inactive, Q continued to rise to 160% of the control values. At 5.3 kPa, oxygen consumption (as determined by the Fick principle) fell to 29% of the normoxic value and was associated with increased branchial vascular resistance (Rg) and an increased diffusion limitation of the gills (Ldiff). Adrenaline increased heart rate and aortic blood pressures. Increased Rg and Ldiff could be provoked in normoxia by the injection of the beta-adrenergic blocking drug propranolol. We suggest that catecholamines may be involved in the tonic control of gill vasomotor tone.

Isolation and characterization of a protein from hagfish serum that is homologous to the third component of the mammalian complement system.

The 192-kDa protein HX, a major component of serum that specifically binds to zymosan particles, was prepared from the plasma of the hagfish (Eptatretus burgeri) by ion-exchange chromatography and gel filtration. HX, present at a concentration of 0.8 mg/ml in the original plasma, was composed of two distinct subunits of 115 kDa and 77 kDa, respectively, which were linked by disulfide bonds. The protein had the same electrophoretic mobility as beta-globulin. Digestion by trypsin resulted in a specific cleavage of the 115-kDa subunit and a change in its immunoelectrophoretic mobility in the anodal direction, leaving the 77-kDa subunit intact. Treatment with SDS and urea resulted in the splitting of the 115-kDa subunits into 68-kDa and 45-kDa components, but this splitting was inhibited by pretreatment with methylamine, suggesting the presence of a thiol ester bond in the 115-kDa subunit. The amino acid composition of HX revealed a striking resemblance to that of human C3. We conclude, therefore, that the 192-kDa protein isolated in this study is analogous to C3, which plays a key role in the mammalian C system.

Immunoreactive endothelin in plasma of nonmammalian vertebrates.

Immunoreactive endothelin (ir-ET) was estimated in plasma of nonmammalian vertebrates, using radioimmunoassay (RIA) for endothelin-1 (ET-1). Blood samples were collected from unanesthetized animals. Plasma ET was determined by RIA after extraction. Plasma levels of ir-ET were 0.7 +/- 0.2 pg/ml (n = 6) in the hagfish Eptatretus burgeri, 4.3 +/- 0.9 pg/ml (n = 5) in the banded dogfish Triakis scyllia, 3.6 +/- 0.8 pg/ml (n = 3) in the common Japanese conger Conger myriaster, 9.6 +/- 1.4 pg/ml (n = 7) in the carp Cyprinus carpio, 6.4 +/- 0.8 pg/ml (n = 5) in the bullfrog Rana catesbeiana, 6.7 +/- 0.6 pg/ml (n = 3) in the soft-shelled turtle Trionyx sinensis japonica, and 3.3 +/- 0.6 pg/ml (n = 8) in the Japanese quail Coturnix coturnix japonica. The dilution curves of the plasma extracts from each species almost paralleled the standard curve for ET-1. Analysis of the plasma extracts of the carp by reverse-phase high-performance liquid chromatography revealed that ir-ET consisted of three components, a predominant peak being located at the elution position of synthetic ET-1. The present results demonstrate clearly that an ET-1-like substance circulates in blood of nonmammalian vertebrates, suggesting an endocrine function of the peptide in these species.

CO2 transport properties of the blood of a primitive vertebrate, Myxine glutinosa (L.).

The CO2 transport properties of the blood of the hagfish, Myxine glutinosa are markedly different from those previously demonstrated for the lamprey, Petromyzon marinus. As in most other vertebrates, the majority of the CO2 in Myxine blood is transported in the form of plasma bicarbonate. Erythrocyte bicarbonate does have some access to the plasma in the blood of Myxine although the documented bicarbonate movements were not sensitive to the chloride/bicarbonate exchange inhibitor, 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS). The transmembrane pH gradient in hagfish erythrocytes is also quite different from the gradient previously demonstrated in other agnathans. As in other agnathans, significant differences do occur between the distribution ratios for protons, chloride and bicarbonate ions across the erythrocyte membrane, although the magnitude of these differences is smaller in Myxine. Finally, the absence of pH-dependent changes in erythrocyte water content appears to be a common feature of agnathan erythrocytes.

Calculation and experimental verification of the frictional ratio of hagfish proteinase inhibitor.

A major structural feature of the alpha-2-macroglobulin-like inhibitor of hagfish described in Osada, Nishigai, and Ikai [(1987) J. Ultrastruct. Mol. Struct. Res. 96, 00-00] was its highly open quaternary structure observed under an electron microscope. We drew a qualitative conclusion that the high frictional ratio obtained from the result of sedimentation study and the large Stokes radius obtained in gel chromatographic experiment were the reflection of such an open quaternary structure. In this paper I present several structural models of hagfish inhibitor based on its electron micrographs and calculate expected frictional ratios for such models according to the method developed by Bloomfield and his co-workers. Their method allows the calculation of frictional coefficient of a body of an arbitrary shape by approximating it with a collection of small spheres. To test the validity of such a method, macroscopic models were built from plastic spheres or cylindrical capsules and their translational frictional coefficients were measured by the free-falling method under experimental conditions where the Reynolds number was between 10(-3) and 10(-4).

Open quaternary structure of the hagfish proteinase inhibitor with similar properties to human alpha-2-macroglobulin.

A homologous protein to human plasma alpha-2-macroglobulin (alpha-2-M) was purified from the blood plasma of hagfish (Eptatretus buergeri) and its structure and function were studied. The hagfish protein inhibited several proteinases and its inhibitory activity was blocked with methylamine as in the case of human alpha-2-M. The molecular weight and sedimentation coefficient of the hagfish inhibitor were 390,000 +/- 20,000 and 11.0 S, respectively, as determined by sedimentation studies. The frictional ratio calculated from these parameters was 1.75. The Stokes radius estimated from HPLC gel chromatography was 8.8-8.9 nm, which was similar to that of human alpha-2-M despite the fact that the hagfish inhibitor was only one-half as large as human alpha-2-M in molecular weight. The hagfish inhibitor was expected to be more asymmetric and/or more hydrated than the human inhibitor. The electron micrographs of the negatively stained hagfish inhibitor showed that it had an open, rectangular quaternary structure of 15 +/- 1.5 X 19 +/- 2 nm in which two semiglobular units were located at the two shorter sides with a gap of 8 +/- 1 nm in width. Each semiglobular unit had an approximate width of 5 +/- 0.5 nm. The thickness of the unit was estimated to be 3 to 3.5 nm from the result of fixed-angle shadowing experiments. Although the two semiglobular units must be connected by some structure, very little material could be seen between them. Such an open quaternary structure may explain the high frictional ratio and large Stokes radius of this protein. The structural change of the inhibitor after reaction with proteinases or methylamine could be detected by electron microscopy and gel chromatography.