Different sensitivities of arteries and veins to vasoactive drugs in a hagfish, Eptatretus cirrhatus

We used myography on five different arteries and three veins of the hagfish, Eptatretus cirrhatus, to test the response of vessels to vasoactive drugs. Concentration-response curves were generated for carbachol, endothelin-1, arginine vasotocin and the adrenergic agonists, phenylephrine and isoprenaline. pEC50 values indicated that veins were more sensitive to endothelin-1 than were arteries, but the arteries were more sensitive to the cholinergic agonist, carbachol. Segmental arteries did not react to arginine vasotocin, but all other vessels did, and on a molar basis it was the most potent agonist tested. That ventral and dorsal aortas were more sensitive to arginine vasotocin than smaller vessels might indicate that this neurohypophysial peptide has the potential to exert a profound influence on branchial vascular resistance and cardiac output in hagfishes. The results also demonstrate the potential for a variety of endogenous peptides to contribute to central venous tone.     

Nucleoside uptake by red blood cells from a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), is mediated by a nitrobenzylthioinosine-insensitive transport system

Red blood cells from the Pacific hagfish (Eptatretus stouti) were found to possess a facilitated diffusion nucleoside transport system insensitive to inhibition by the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). Uridine uptake by this route was saturable (apparent Km 0.14 mM; Vmax 2 mmol/l cells per h at 10 degrees C), inhibited by inosine and adenosine, and blocked both by the vasodilator dipyridamole and by the thiol-reactive agent p-chloromercuriphenylsulphonate. The properties of this carrier resemble closely those of NBMPR-insensitive nucleoside transport systems in some mammalian neoplastic cell lines and in rat red cells. The presence of this type of carrier in a primitive vertebrate suggests that such transporters have a broad biological distribution and that they pre-date or arose at an early stage of vertebrate evolution.     

Distinct Ig H chains in a primitive vertebrate, Eptatretus stouti

Serum Ig from the Pacific hagfish, Eptatretus stouti, was isolated by affinity chromatography using a specific mAb (H.45). Analysis of the approximately 210-kDa molecule by SDS-PAGE under reducing conditions revealed two H chains of approximately 77 kDa (H1) and approximately 70 kDa (H2) and L chains of approximately 30 kDa. H1 and H2 were shown to differ with respect to their peptide maps, amino-terminal amino acid sequences, and reactivity to the mAb H.45, suggesting that they represent discrete H chain isotypes. Two-dimensional nonreducing/reducing SDS-PAGE demonstrated that H and L chains were covalently linked predominantly as H-H-L and H-L configurations. Noncovalently bound L chains were also found. H-H-L complexes were shown to contain H1-H2 heterodimers of H chains in addition to H1-H1 homodimers.     

Complement-like protein from the phylogenetically primitive vertebrate,Eptatretus Stouti,is a Humoral Opsonin

• 1.1. Serum from the Pacific hagfish,Eptatretus stouti,contains a complement-like protein (CLP).
• 2.2. CLP from unfractionated hagfish serum and from affinity-purified preparations binds to yeast cell surfaces.
• 3.3. Incubation with CLP enhances the phagocytosis of yeast by hagfish leukocytes.
• 4.4. CLP-mediated opsonization can be inhibited by anti-CLP antibody, EDTA, d(+)mannose and l(+)rhamnose.
• 5.5. Additional opsomic factors are also in hagfish serum.

     

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.     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

Structure and function of the velar muscle in the New Zealand hagfish Eptatretus cirrhatus: response to temperature change and hypoxia

The rate of velar movement in Eptatretus cirrhatus, as determined by electromyography, increased with Q(10) 3·2 during exposure to temperatures between 7 and 19° C and increased 3·9 fold during exposure to hypoxia (oxygen partial pressure = 6·67 kPa). This confirms the role of the velum in generating respiratory currents and modification of its activity in response to changes in metabolic demand or environmental oxygen availability. The maximum velar rate observed was 168 beats min(-1) , higher than that recorded in any hagfish species to date. Fibres of musculus craniovelaris were exclusively small, red (slow-twitch) fibres, consistent with a high aerobic capacity required by fibres involved in rhythmic, ongoing activity.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Conformational changes in phosphoglucose isomerase induced by ligand binding

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is the second enzyme in glycolysis, where it catalyzes the isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate. It is the same protein as autocrine motility factor, differentiation and maturation mediator, and neuroleukin. Here, we report a new X-ray crystal structure of rabbit PGI (rPGI) without ligands bound in its active site. The structure was solved at 1.8A resolution by isomorphous phasing with a previously solved X-ray crystal structure of the rPGI dimer containing 6-phosphogluconate in its active site. Comparison of the new structure to previously reported structures enables identification of conformational changes that occur during binding of substrate or inhibitor molecules. Ligand binding causes an induced fit of regions containing amino acid residues 209-215, 245-259 and 385-389. This conformational change differs from the change previously reported to occur between the ring-opening and isomerization steps, in which the helix containing residues 513-521 moves toward the bound substrate. Differences between the liganded and unliganded structures are limited to the region within and close to the active-site pocket.     

The partial sequence of the first 30 residues from the amino-terminus of hemoglobin B in the hagfish,Eptatretus stoutii: Homology with lamprey hemoglobin

Summary The partial sequence of the first 30 residues from the amino-terminus of hemoglobin B in the hagfish,Eptatretus stoutii, has been determined. Considerable homology was found between this sequence and the corresponding sequence of lamprey hemoglobin. Both hagfish and lamprey hemoglobins have an additional segment of 9 residues at the amino-terminus as compared with mammalian hemoglobins.This work was initiated at the University of Texas at Austin, and continued in Dr. Charles Yanofskey's laboratory at Stanford University.     

Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish,Eptatretus burgeri

Summary Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. These types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.We wish to express our appreciation to Professor Hideshi Kobayashi for the guidance of this study. It was aided by grants from the Ford Foundation and the Ministry of Education to Professor H. Kobayashi, and also by grants from the Population Council, New York and the Ministry of Education to Professor S. Ishii.     

The high intracellular ph buffering of hagfish (Eptatretus cirrhatus) dental plate retractor muscle cannot be attributed to the known histidine-related compounds present in other vertebrates

• 1.1. Intracellular pH buffering capacity of hagfish (Eplatretus cirrhatus) dental plate retractor muscles is among the highest reported for any vertebrate muscle.
• 2.2. Over 80% of the pH buffering capacity of hagfish retractor and myotome muscle is due to components other than proteins and phosphate.
• 3.3. The muscles have less than 0.5 μmol/g wet weight of l-histidine, and lack l-l-methyl histidine, l-3-methyl histidine and the histidine-containing dipeptides anserine, carnosine and ophidine.
• 4.4. Instead, they contain an unidentified low molecular weight acid-soluble compound to which the high pH buffering capacity can be attributed.

     

NO binding induced conformational changes in a truncated hemoglobin from Mycobacterium tuberculosis

The resonance Raman spectra of the NO-bound ferric derivatives of wild-type HbN and the B10 Tyr --> Phe mutant of HbN, a hemoglobin from Mycobacterium tuberculosis, were examined with both Soret and UV excitation. The Fe-N-O stretching and bending modes of the NO derivative of the wild-type protein were tentatively assigned at 591 and 579 cm(-1), respectively. Upon B10 mutation, the Fe-NO stretching mode was slightly enhanced and the bending mode diminished in amplitude. In addition, the N-O stretching mode shifted from 1914 to 1908 cm(-1). These data suggest that the B10 Tyr forms an H-bond(s) with the heme-bound NO and causes it to bend in the wild-type protein. To further investigate the interaction between the B10 Tyr and the heme-bound NO, we examined the UV Raman spectrum of the B10 Tyr by subtracting the B10 mutant spectrum from the wild-type spectrum. It was found that, upon NO binding to the ferric protein, the Y(8a) mode of the B10 Tyr shifted from 1616 to 1622 cm(-1), confirming a direct interaction between the B10 Tyr and the heme-bound NO. Furthermore, the Y(8a) mode of the other two Tyr residues at positions 16 and 72 that are remote from the heme was also affected by NO binding, suggesting that NO binding to the distal site of the heme triggers a large-scale conformational change that propagates through the pre-F helix loop to the E and B helices. This large-scale conformational change triggered by NO binding may play an important role in regulating the ligand binding properties and/or the chemical reactivity of HbN.     

Crystallographic analysis of synechocystis cyanoglobin reveals the structural changes accompanying ligand binding in a hexacoordinate hemoglobin

The crystal structures of cyanide and azide-bound forms of the truncated hemoglobin from Synechocystis are presented at 1.8 angstroms resolution. A comparison with the structure of the endogenously liganded protein reveals a conformational shift unprecedented in hemoglobins, and provides the first picture of a hexacoordinate hemoglobin in both the bis-histidyl and the exogenously coordinated states. The structural changes between the different conformations are confined to two regions of the protein; the B helix, and the E helix, including the EF loop. A molecular "hinge" controlling movement of the E helix is observed in the EF loop, which is composed of three principal structural elements: Arg64, the heme-d-propionate, and a three-residue extension of the F helix. Additional features of the structural transition between the two protein conformations are discussed as they relate to the complex ligand-binding behavior observed in hexacoordinate hemoglobins, and the potential physiological function of this class of proteins.     

Distribution of 5-HT (serotonin) immunoreactivity in the central nervous system of the inshore hagfish,Eptatretus burgeri

Summary Immunohistochemical techniques were used to study the distribution of serotonin in the central nervous system of the hagfish,Eptatretus burgeri, in order to produce a detailed map of serotonin-containing structures. In the hypothalamus, many serotonin-containing neurons contacted the cerebrospinal fluid. Most of the serotonin-containing cell bodies were located in the raphe region, where they were compactly distributed at the level of the nucleus motorius tegmenti pars anterior but more diffusely distributed at the level of the nucleus motorius tegmenti pars posterior. Serotonin-containing cell bodies and varicose fibers were widely distributed throughout the brain and upper spinal cord segments, but the distribution density was not even. On the basis of its abundance, serotonin can be judged to have an important function in the control of the hagfish central nervous system. From a phylogenetic point of view, serotonin-containing neurons in the raphe region appear to be a common property of all classes of vertebrates studied except the lampreys, whereas serotonin-containing cerebrospinal fluid-contacting neurons may be considered to be a primitive condition in all nonmammalian vertebrates.     

FMRFamide-like immunoreactivity in the brain of the Pacific hagfish,Eptatretus stouti (Myxinoidea)

Summary The distribution of FMRFamide-like immunoreactivity was investigated in the brain of a myxinoid, the Pacific hagfish,Eptatretus stouti, by means of immunocytochemistry. In the forebrain, labelled cell bodies occurred in the infundibular nucleus of the hypothalamus and some closely adjacent nuclei. Labelled fibers formed a diffuse network in the forebrain, but there was no evidence for the presence of intracerebral ganglionic cells of the terminal nerve or a central projection of the terminal nerve. In the hindbrain, a group of labelled cells was found in the trigeminal sensory nucleus. A distinet terminal arborization occurred in the ventrally adjacent nucleus A of Kusunoki and around the nuclei of the branchial motor column. These findings suggest that FMRFamide may play a role in the central control of branchiomotor activity.     

Immunocytochemical localization of somatostatin and vasotocin in the brain of the Pacific hagfish,Eptatretus stouti

Summary The brain of the Pacific hagfish, Eptatretus stouti, was studied immunocytochemically using antisera against somatostatin (SRIH), arginine vasopressin (AVP), and adrenocorticotropic hormone (ACTH). SRIH-immunoreactive perikarya were distributed bilaterally in the postoptic nucleus and in the hypothalamic nucleus. Although several short, stained fibers were observed in the vicinity of the perikarya, SRIH-immunoreactivity was not found in the neurohypophysis, nor in other parts of the brain. On the other hand, presumed arginine vasotocin (AVT) perikarya were distributed in an arc-shaped region extending from the posterior part of the preoptic nucleus to the anterior-most end of the hypothalamic nucleus and projected their fibers to the neurohypophysis. Most presumptive AVT perikarya were located close to the paired prehypophysial arteries near the anterior end of the postoptic nucleus. In the neurohypophysis, abundant presumptive AVT-fibers terminated in the posterior dorsal wall, although some fibers terminated in the anterior dorsal wall and only a few fiber endings were found in the ventral wall. No ACTH-positive cells were detected in the hagfish brain or in the pituitary gland.Supported from a grant from the National Science Foundation PCM 8141393