Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.     

Cerebrospinal fluid contacting neurons in the reduced brain ventricular system of the atlantic hagfish,Myxine glutinosa

Cerebrospinal fluid (CSF)-contacting neurons are sensory-type cells sending ciliated dendritic process into the CSF. Some of the prosencephalic CSF-contacting neurons of higher vertebrates were postulated to be chemoreceptors detecting the chemical composition of the CSF, other cells may percieve light as "deep encephalic photoreceptors". In our earlier works, CSF-contacting neurons of the mechanoreceptor-type were described around the central canal of the hagfish spinal cord. It was supposed that perceiving the flow of the CSF they are involved in vasoregulatory mechanisms of the nervous tissue. In the present work, we examined the brain ventricular system of the Atlantic hagfish with special reference to the presence and fine structure of CSF-contacting neurons. Myxinoids have an ontogenetically reduced brain ventricular system. In the adult hagfish (Myxine glutinosa) the lumen of the lateral ventricle is closed, the third ventricle has a preoptic-, infundibular and subhabenular part that are not connected to each other. The choroid plexus is absent. The infundibular part of the third ventricle has a medial hypophyseal recess and, more caudally, a paired lateral recess. We found CSF-contacting neurons in the lower part of the third ventricle, in the preoptic and infundibular recess as well as in the lateral infundibular recesses. No CSF-contacting neurons were found in the cerebral aqueduct connecting the subhabenular recess to the fourth ventricle. There is a pineal recess and a well-developed subcommissural organ at the rostral end of the aqueduct. Extending from the caudal part of the fourth ventricle in the medulla to the caudal end of the spinal cord, the central canal has a dorsal and ventral part. Dendrites of CSF-contacting neurons are protruding into the ventral lumen. Corroborating the supposed choroid plexus-like function of the wall of the dorsal central canal, segmental vessels reach a thin area on both sides of the ependymal lining. The perikarya of the CSF-contacting neurons found in the brain ventricles are mainly bipolar and contain granular vesicles of various size. The bulb-like terminal of their ventricular dendrites bears several stereocilia and contains basal bodies as well as mitochondria. Basal bodies emit cilia of the 9+0-type. Cilia may arise from the basal body and accessory basal body as well. The axons run ependymofugally and enter--partially cross--the periventricular synaptic zones. No neurohemal terminals similar to those formed by spinal CSF-contacting neurons of higher vertebrates have been found in the hagfish. We suppose that CSF-contacting neurons transform CSF-mediated non-synaptic information taken up by their ventricular dendrites to synaptic one. A light-sensitive role for some (preoptic?) groups of CSF-contacting neurons cannot be excluded.     

Immunolocalization of Na+/K(+)-ATPase in mitochondrion-rich cells of the atlantic hagfish (Myxine glutinosa) gill

Using a monoclonal antibody for the alpha-subunit of the Na+/K(+)-ATPase, DASPEI (a vital mitochondria dye), and confocal laser scanning microscopy, the presence of Na+/K(+)-ATPase in mitochondrion-rich cells of the hagfish gill was confirmed. In addition, the level of Na+/K(+)-ATPase expression in the hagfish gill was compared to that of fishes with different osmoregulatory strategies (little skate, Raja erinacea and mummichog, Fundulus heteroclitus). Immunocytochemistry detected a high density of columnar cells expressing Na+/K(+)-ATPase in the afferent filamental epithelium. Positive cells were also found in the lamellar epithelium but at a much lower density. The distribution of DASPEI staining was similar to that of the Na+/K(+)-ATPase antibody, indicating that the enzyme is expressed in mitochondrion-rich cells. Immunoblot analysis confirmed the specificity of the antibody for the 97 kDa alpha-subunit of the enzyme. The immunoreactive band intensity for the Atlantic hagfish was similar to that of the little skate, but less than half that of the full-strength seawater mummichog. These results are discussed in relation to gill function in early craniates.     

The fine structure of the kidney of the hagfish (Myxine glutinosa L.)

Summary The fine structure of the kidney (glomerulus and archinephric duct) of the hagfish, Myxine glutinosa (L.) was studied in thin sections and by freeze-fracture technique.The glomerular filtration barrier is similar to that of mammalian kidneys. However, endothelial fenestrations are relatively scanty and the basement membranes of endothelial cells and podocytes always appear separated by a layer of collagen fibrils and microfibrils often surrounding numerous and extended mesangial cells. Between podocytes and their processes maculae occludentes and peculiar junctions of another type occur.The zonulae occludentes between epithelial cells of the archinephric duct are composed of five or more strands, occasionally of only one or two.Supported in part by Deutsche Forschungsgemeinschaft (SFB 146, STO B4) and NIH (ISOI-RR 05764).We are grateful to Dipl.-Ing. G. Wermbter for her helpful criticism and to Mr. H. Heidreich for his excellent technical assistance.     

Ultrastructure of four types of striated muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary Four types of striated muscle fibers with distinctive ultrastructure were defined in the Atlantic hagfish (Myxine glutinosa, L.): white, intermediate, and red fibers of m. parietalis, and red fibers of m. craniovelaris.White fibers are thick, contain very few mitochondria and fat vacuoles, and possess distinct and separate myofibrils with thin Z-disks and distinct M-lines. Intermediate fibers are thinner, possess largely similar myofibrils that often are even better separated due to a higher content of fat vacuoles and especially mitochondria and glycogen granules. Red fibers of m. parietalis contain large amounts of mitochondria, fat vacuoles, and glycogen granules. Their myofibrils possess M-lines, and although branching more, the myofibrils of red fibers conform with a Fibrillenstruktur pattern like those of white and intermediate fibers. Red fibers of m. craniovelaris are very thin, possess many smaller fat vacuoles, and large amounts of mitochondria and glycogen granules. The myofibrils are significantly thinner than in m. parietalis fibers, run as quite independent well separated units, possess thicker Z-disks, and lack M-lines. Large amounts of myosatellite cells are associated with these red fibers.Triads are located near A/I-junctions in all four fiber types and occur irregularly, the density of triads being different in the various fiber types.We are indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and we also wish to thank Dr. Jan K. S. Jansen, Institute of Physiology, University of Oslo, for valuable suggestions during this study.     

Glomerular bypass shunts in the kidney of the Atlantic hagfish,Myxine glutinosa

Summary The vascular pathways associated with the glomerulus of the Atlantic hagfish, Myxine glutinosa have been studied by scanning electron microscopy of corroded resin casts of the vasculature. Although the overall pattern of the renal vasculature did not differ from earlier reports, a previously unreported vascular pathway which arose from the renal artery and bypassed the glomerular capillaries in 28% of glomeruli was clearly demonstrated. Glomerular bypass shunts either ran to join the loose capillary network around Bowman's capsule and thereby drain into the network of vessels associated with the mesonephric duct (ureter), or ran directly into the ureteral system of vessels and subsequently into the posterior cardinal veins. Glomerular bypass shunts which theoretically permit renal arterial blood to bypass the process of filtration may play a role in the regulation of body fluid volume.     

The semicircular canals of the hagfish Myxine glutinosa

The internal radius (r) and radius of curvature (R) of the single semicircular canals of Myxine glutinosa have unusual dimensions. In mammals and fish the increase in dimension of r and R with respect to body weight is small; in fish r is larger than in mammals of equivalent weight in order to increase the sensitivity of the canals to angular rotation and R increases correspondingly (Jones & Spells, 1963). In Myxine r is larger than in fish or mammals yet R is smaller. It is argued that the large internal radius is the result of the need to increase the sensitivity of a single canal which has to signal rotation in three planes while the small radius of curvature follows from the absence of a cupula. In order to verify that the cristae of the canals do respond to rotational velocity, recordings were obtained from the nerves serving the canals during rotation in the horizontal plane. The frequency response of several afferents recorded simultaneously at sinusoidal rotations between 0.25 and 2.0 Hz was in the form of a sine wave 90 in advance of head position, as would be expected of a velocity transducer. The gain of single afferents was an order of magnitude less than those reported for other vertebrates.     

Binding sites for atrial natriuretic peptide in the kidney and aorta of the hagfish (Myxine glutinosa)

1. In the atlantic hagfish (Myxine glutinosa) binding sites for atrial natriuretic peptide (ANP) were visualized by autoradiography in contractile structures of the renal system (glomeruli, neck segment, and archinephric duct) and in the aorta. 2. Since the location of binding sites is comparable to that in higher vertebrates including man, these results suggest that ANP may act as a hormone already in cylcostomata.     

Cell types in the adenohypophysis of the hagfish,Myxine glutinosa (Cyclostomata)

Summary The anterior pituitary of the Atlantic hagfish, Myxine glutinosa, was investigated by transmission electron microscopy. The cells of the adenohypophysis are arranged in follicles surrounded by connective tissue. Five cell types containing granules and one agranular cell type were identified. At the present state of the study the function of these cells remains open to discussion.This study was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (Grant no. 4070) and the Stiftungs- und Förderungsgesellschaft der Paris Lodron-Universität in Salzburg. The authors are grateful to the Director of the Biological Station in Drøbak (Norway), Amanuensis Finn Walvig, for procuring the material     

Plasma cells in adult Atlantic hagfish,Myxine glutinosa

Summary Hagfishes, the most primitive vertebrates, are of special interest for the evolution of immune responses. Eptatretus stoutii, the Pacific hagfish, is able to mount cellular and humoral immune responses but all attempts to demonstrate in them the presence of plasma cells have failed. In the present study we demonstrate for the first time plasma cells identifiable by ultrastructural criteria in the pronephros, a primitive lymphohaemopoietic organ, of Myxine glutinosa, the Atlantic hagfish.     

Ultrastructure of motor nerve terminals on different types of muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary White and intermediate parietal muscle fibers of Myxine are innervated focally at one end. Most synaptic vesicles are empty. These terminals also contain 1–2% large 800–1.100 Å dense-core vesicles. Red fibers of parietal and craniovelar muscle are innervated in a distributed fashion, and the presynaptic profiles contain a higher number of large dense-core vesicles (averaging 9% and 15%, respectively; up to 37%). For all terminals the synaptic gap is 450–600 Å wide, and postsynaptic folds are absent.Empty synaptic vesicles exist as round or elongated profiles. The proportion of elongated profiles increases by formation from round ones when increasing the molarity of the buffer in the aldehyde fixative. Furthermore, the proportion of elongated vesicle profiles in terminals on Myxine white fibers at different buffer molarities, is identical with that in mammalian motor terminals at similar molarities. On this basis the significance and mode of formation of elongated vesicle profiles is discussed. The conclusion is made that the susceptibility of flattening depends on the osmotic pressure of the vesicle contents once the aldehyde has influenced the vesicle membrane.The different vesicle populations in terminals on different types of muscle fibers are significant. Terminals on red fibers probably contain serotonin (5-HT) either as sole transmitter or in addition to acetylcholine.The author is indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and to Mrs. Jorunn Line Vaaland for expert technical assistance.     

Fine structure of the agranular adenohypophysial cells in the hagfish,Myxine glutinosa (Cyclostomata)

Summary Agranular cells are found throughout the adenohypophysis of Myxine glutinosa. They are located at the margin of single hypophysial follicles in the form of a spongy epithelial net. Their relationship to the secretory cells of the adenohypophysis and their morphology are described in adult animals. The distribution and ultrastructure of the agranular cells in the hagfish are in good agreement with the pattern of comparable cells in other vertebrates.This study was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (grant no. 3204 and 3494). The authors are grateful to the director of the Biological Station in Drøbak (Norway), Amanuensis Finn Walvig, for procuring the material, to Miss Synnøve Tholo for technical assistance and to Mr. Arno Laminger for photographic work     

Vascular bed-specific endothelium-dependent vasomomotor relaxation in the hagfish, Myxine glutinosa

The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates that lived some time more than 500 million years ago. Features that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. We recently reported that hagfish endothelium displays phenotypic heterogeneity in ultrastructure, lectin binding, and mechanisms of leukocyte adhesion. Thus, phenotypic cell heterogeneity evolved as an early feature of the endothelium. In the present study, we wanted to extend these observations by determining whether hagfish endothelium plays a role in mediating vasomotor tone. Response of mesenteric and skeletal muscle arteries to a variety of mediators was assayed by videomicroscopy. Phenylephrine and acetylcholine induced vasoconstriction of mesenteric and skeletal muscle arteries. Bradykinin (BK) and ADP promoted vasorelaxation in precontracted mesenteric arteries but not those from skeletal muscle. BK- and ADP-mediated vasorelaxation of the mesenteric artery was abrogated by mechanical denudation of the endothelium but was unaffected by N(G)-nitro-L-arginine methyl ester. Indomethacin significantly inhibited the vasodilatory response to ADP but not BK. The nitric oxide donor sodium nitroprusside resulted in endothelium-independent relaxation of both mesenteric and skeletal muscle arteries. Together, these data suggest that site-specific endothelium-dependent vasorelaxation is an evolutionarily conserved property of this cell lineage.     

Endocytosis in the ureteric duct epithelium of the hagfish (Myxine glutinosa L.)

Summary Solutions containing ferritin or thorotrast particles were microperfused through the ureteric duct of the hagfish. The markers were taken up by the epithelial cells by way of endocytosis and were transported in bulk in apical vesicles. Newly formed apical vesicles containing marker showed bristle coating on the cytoplasmic side of their limiting membrane. This coating appeared to be lost during the movement of vesicles deeper into the cytoplasm.The findings indicate that the epithelial cells in the ureteric duct have capablity for extensive bulk uptake of macromolecules from the luminal fluid. The mechanisms involved in absorption appear to be similar to those in proximal convoluted tubules of mammals.The apical dense tubules observed with some fixation techniques appear to represent collapsed endocytotic vesicles.The authors are indebted to Finn Walwig, Cand. real., Marine Biology Station, University of Oslo, Dröbak, Norway for kindly supplying the hagfishes used in this study. The technical assistance of Miss Signe Fjeldsenden and Miss Britt-Marie Pettersson is gratefully acknowledged.This work has been supported by grants from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne).     

Intercellular junctions in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary The intramembrane organization of the occluding junctions in the gill epithelium of the Atlantic hagfish, Myxine glutinosa, was studied by means of freeze-fracture electron microscopy. Mitochondria-rich cells, characterized by assemblies of rod-shaped particles in the luminal plasma membrane and by an extensive intracellular amplification of the basolateral plasma membrane, are singly distributed between the pavement cells in the gill epithelium of this marine and stenohaline cyclostome. The occluding junctions between mitochondria-rich cells and pavement cells do not differ from those between adjacent pavement cells, concerning the number of superimposed strands (median 6, range 4–9) and their geometrical organization. These observations suggest that, in contrast to marine teleosts, the paracellular pathway plays a minor role in transepithelial ion movements in the hagfish gill epithelium. The findings are in agreement with the absence of hypoosmoregulatory mechanisms in hagfish, as have been evolved in various marine vertebrates. In addition, small communicating junctions are demonstrated between pavement cells; they possibly serve for a coordinated synthesis and secretion of mucus by the pavement cells.Dedicated to Prof. H. Leonhardt on the occasion of his 70th birthday     

Velocity and displacement receptors in the skin of the hagfish Myxine glutinosa.

The skin of Myxine glutinosn contains both velocity-sensitive and displacement-sensitive mechanoreceptors. These can be distinguished from each other by the different time course of their response to skin indentation. Velocity receptors gave a brief response, consisting usually of three to four action potentials which could only be elicited by movement of the skin. No velocity receptor was spontaneously active. Displacement receptors, some of which were spontaneously active, gave a prolonged discharge which continued, with only slight adaptation, while the skin was indented. Velocity receptors code for velocity of indentation and continue to respond to repeated stimuli. Displacement receptor action potentials occur irregularly, with a Poisson-like distribution of interspike interval. The mean frequency of displacement receptor discharge increases with depth of skin indentation, while the interspike interval distribution becomes progressively biased towards shorter intervals. The threshold of displacement receptors is related to displacement velocity; sinusoidal indentations of the skin produce a minimum threshold around 60 Hz. These receptors fail to respond to oscillations in excess of 120 Hz. The structure of the skin is described. Stress-strain curves are given both for static and dynamic compression of the skin and these are discussed in relation to the properties of the skin mechanoreceptors.     

Presence of IGF-1-like peptides in the neuroendocrine system of the Atlantic hagfish, Myxine glutinosa (Cyclostomata): evidence derived by chromatography, radioimmunoassay and immunohistochemistry

By the use of radioimmunoassay and chromatography peptides related to insulin-like growth factor 1 (IGF-1) have been identified in the cylostomian species Myxine glutinosa. IGF-1-like-immunoreactivity was detected in serum as well as in brain, intestine, pancreas and liver. After acid gel chromatography, the IGF-1-like immunoreactivity eluted as one major peak, with an apparent molecular weight of between 2-4 kDa. When the same antiserum was applied immunohistochemically, IGF-1-like-immunoreactivity was observed in endocrine cells of the mucosal epithelium throughout the primitive intestinal tube. These cells were of the open type and occurred in small clusters. In addition, the majority of the endocrine cells of the pancreas of Myxine displayed IGF-1-like-immunoreactivity. In some of the specimens investigated IGF-1-like-immunoreactive perikarya and fibers were observed on all levels of the brain. Distribution patterns and densities of the IGF-1-like-immunoreactive structures in Myxine correlated with the measurements obtained by radioimmunoassay. Absorption studies with insulin- and IGF-related peptides as well as with crude extracts and the peak material obtained after gel chromatography indicated that the IGF-1-like peptides in Myxine are different from mammalian and non-mammalian insulins as well as from mammalian IGF-1. Generally, the results suggest a long phylogenetic history of IGF-1-like peptides and indicate their fundamental functional impact in all vertebrates.