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Structure and expression of three Emx genes in the dogfish Scyliorhinus canicula: functional and evolutionary implications.

We report the characterization of three Emx genes in a chondrichthyan, the dogfish Scyliorhinus canicula. Comparisons of these genes with their osteichthyan counterparts indicate that the gnathostome Emx genes belong to three distinct orthology classes, each containing one of the dogfish genes and either the tetrapod Emx1 genes (Emx1 class), the osteichthyan Emx2 genes (Emx2 class) or the zebrafish Emx1 gene (Emx3 class). While the three classes could be retrieved from the pufferfish genome data, no indication of an Emx3-related gene in tetrapods could be found in the databases, suggesting that this class may have been lost in this taxon. Expression pattern comparisons of the three dogfish Emx genes and their osteichthyan counterparts indicate that not only telencephalic, but also diencephalic Emx expression territories are highly conserved among gnathostomes. In particular, all gnathostomes share an early, dynamic phase of Emx expression, spanning presumptive dorsal diencephalic territories, which involves Emx3 in the dogfish, but another orthology class, Emx2, in tetrapods. In addition, the dogfish Emx2 gene shows a highly specific expression domain in the cephalic paraxial mesoderm from the end of gastrulation and throughout neurulation, which suggests a role in the segmentation of the cephalic mesoderm.

Amino Acid Sequence↗

The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias).

The aim of the present study was to evaluate the effects of endothelin-l-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg(-1)) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O2. The effects of endothelin-1 on arterial blood partial pressure of CO2 were not synchronised with the changes in O2 partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO2 partial pressure was increased transiently by approximately 1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO2 partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (approximately 0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml(-1)) eliminated the increase in CO2 partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO2 partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by approximately 10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.

Animals↗

The involvement of melanotrophins in physiological colour change in the dogfish Scyliorhinus canicula.

Desacetylated alpha-, beta-, and gamma-MSH purified from the neurointermediate lobe of the pituitary gland of the dogfish Squalus acanthias were all equipotent in turning partially hypophysectomized dogfish, Scyliorhinus canicula, dark. However, each possessed less than 2% of the melanotrophic activity of either acetylated mammalian alpha-MSH or synthetic acetylated Squ. acanthias alpha-MSH. These results suggest that acetylation is much more important than amidation in determining melanotrophic potency in vivo in the dogfish. Intravenous administration into grey-adapted dogfish of an antibody capable of binding the alpha-MSH-like peptides present in the blood was very effective at inducing pallor. In contrast, a gamma-MSH antibody had no effect on the melanophores of Scy. canicula. Overall the results suggest that it is alpha-melanotrophins that regulate physiological colour change in the dogfish Scy. canicula.

Acetylation↗

A comparison of dogfish and porcine pancreatic lipases.

1. A 16-fold purification of lipase activity from the pancreas of the dogfish (Squalus acanthias) was obtained by preparative isoelectric focussing. 2. The dogfish enzyme has a higher pH optimum (8.5) and a broader spectrum of activity above and below its optimum than a commercial porcine lipase preparation (optimum, 7.5). 3. The temperature optimum of dogfish lipase was 35 degrees C and it displayed greater stability than the porcine lipase preparation towards higher temperatures in the absence of substrate. 4. Dogfish lipase was active against a broad range of triacylglycerols and a wax ester. It had greater relative activity against the latter than the porcine lipase but hydrolyzed low mol. wt fatty acids less readily. In general, the effect of temperature was less on the dogfish enzyme than the porcine one.

Animals↗

Comparison of the bioaccumulation from seawater and depuration of heavy metals and radionuclides in the spotted dogfish Scyliorhinus canicula (Chondrichthys) and the turbot Psetta maxima (Actinopterygii: Teleostei).

The bioaccumulation of selected heavy metals and radionuclides ((241)Am, (109)Cd, (57)Co, (51)Cr, (134)Cs, (54)Mn and (65)Zn) from seawater was experimentally compared in the Chondrichthyan Scyliorhinus canicula (spotted dogfish) and the Actinopterygian Teleost Psetta maxima (turbot), of comparable size, age and benthic feeding habits. The speciation of these elements in seawater (salinity 38 per thousand, pH 8.1, temperature 16.5 degrees C) was also calculated to determine their potential bioavailability. The uptake rates, measured over 14 days, varied greatly among isotopes and between species. Concentration factors (CFs) in P. maxima varied 5-fold between ca. 0.2 for (51)Cr and 2.5 for (65)Zn and (134)Cs, whereas in S. canicula they varied by a much greater factor of 350, with CFs for (51)Cr and (241)Am ranging from ca. 0.4 to 140, respectively. With the exception of (134)Cs, all radiotracers were accumulated at a faster rate in S. canicula than in P. maxima, particularly for (241)Am and (65)Zn where the CFs attained during the uptake phase were, two and one order of magnitude greater in S. canicula, respectively. In contrast, (134)Cs reached a CF of about 2.5 in P. maxima, which was 5-fold greater than in S. canicula. Patterns of loss from the experimental depuration phase over 29 days showed greater similarities between species, compared to the uptake phase that highlighted the greater differences between elements. The distributions of these seven radioisotopes among six body components indicated that between the two species the skin of the dogfish displayed a greater bioaccumulation potential, particularly for (241)Am, (57)Co and (65)Zn. However (65)Zn was also distinctive from (241)Am and (57)Co in its pattern of bioaccumulation in dogfish, with its other body components attaining concentrations of (65)Zn that were comparable to the levels found in its skin. The heightened uptake of (134)Cs in turbot was characterised by a more even percentage distribution among its tissues compared to (241)Am and (57)Co, but every tissue of turbot had a higher concentration of (134)Cs, compared to dogfish, particularly the muscle and liver. The elevated uptake rates and higher CFs for most radioisotopes indicate that S. canicula is more susceptible than P. maxima to exposure and contamination by these metals and radionuclides in seawater. These experimentally-determined differences between dogfish and turbot in their bioaccumulation characteristics were assessed against a set of criteria erected to evaluate the working hypothesis that they were taxonomically based. The outcomes of this initial assessment were supportive of this hypothesis that warrants further investigation.

Animals↗

Development of catecholaminergic systems in the spinal cord of the dogfish Scyliorhinus canicula (Elasmobranchs).

The development of catecholamine-synthesizing cells and fibers in the spinal cord of dogfish (Scyliorhinus canicula L.) was studied by means of immunohistochemistry using antibodies against tyrosine hydroxylase (TH). The only TH-immunoreactive (TH-ir) cells already present in the spinal cord of stage 26 embryos were of cerebrospinal fluid-contacting (CSF-c) type. These cells were the first catecholaminergic neurons of the dogfish CNS. The number of these TH-ir cells increased very considerably in later embryos and adult dogfish. In later embryos (stage 33; prehatching), faintly TH-ir non-CSF-contacting neurons were observed in the ventral horn throughout most of the spinal cord. In adult dogfish, some non-CSF-contacting TH-ir cells were observed ventral or lateral to the central canal. In the rostral spinal cord, the catecholaminergic neurons observed in dorsal regions were continuous with caudal rhombencephalic populations. Numerous TH-ir fibers were observed in the spinal cord of later embryos and in adults, both intrinsic and descending from the brain, innervating many regions of the cord including the dorsal and ventral horns. In addition, some TH-ir fibers innervated the marginal nucleus of the spinal cord. The early appearance of catecholaminergic cells and fibers in the embryonic spinal cord of the dogfish, and the large number of these elements observed in adults, suggests an important role for catecholamines through development and adulthood in sensory and motor functions.

Animals↗

Amino acid sequence of dogfish trypsin.

The amino acid sequence of pancreatic trypsin from the spiny Pacific dogfish (Squalus acanthias) has been determined and compared with the sequences of bovine and porcine trypsin. Dogfish trypsin contains one less amino acid residue (222) than the other two enzymes. Two-thirds of the residues in corresponding positions in dogfish and bovine trypsin are identical and the sequences ofall three enzymes are homologous. Of the 223 amino acid residues of bovine trypsin, 77 are replaced without significant changes in function. Seven replacements, all conservative, occur in the interior of the protein; the remainder are on the surface. All residues known to be components of the active site of bovine trypsin are present in corresponding positions in dogfish trypsin. Comparison of the three enzymes suggests calcium binding sites in dogfish trypsin. A corrected sequence of bovine trypsin identifies residue 67 as Asn and residues 84-87 as Ser-Asn-Thr-Leu.

Amino Acid Sequence↗

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.

Animals↗

Distinct sequence of gonadotropin-releasing hormone (GnRH) in dogfish brain provides insight into GnRH evolution.

In vertebrates, gonadotropin-releasing hormone (GnRH) belongs to a family of decapeptides characterized by the conservation of residues 1, 2, 4, 9, and 10. In the jawed vertebrates only positions 5, 7, and 8 in the GnRH molecules vary. We have now purified two forms of GnRH from the brains of spiny dogfish (Squalus acanthias) by using reverse-phase high-performance liquid chromatography. The primary structures were established by automated Edman degradation and mass spectral analysis. The distinct structure of the first form (dogfish GnRH) is pGlu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-NH2 (pGlu represents pyroglutamyl). The second peptide is identical to a form of GnRH originally isolated from chicken brains (chicken GnRH-II; pGlu-His-Trp-Ser-His-Gly-Trp-Tyr- Pro-Gly-NH2) and is widespread throughout the vertebrates. We are aware of no other species of cartilaginous fish in which the primary structures of two forms of GnRH have been determined. The presence of chicken GnRH-II in dogfish supports the idea that chicken GnRH-II is the oldest GnRH to evolve in jawed vertebrates. With the addition of the dogfish GnRH structure to the family, two main structural branches of GnRH can be delineated. The physiological effects of dogfish GnRH included the release of not only gonadotropin but also growth hormone from goldfish pituitary fragments.

Amino Acid Sequence↗

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Vascular volume-distensibility characteristics of the isolated dogfish gut.

The vascular capacitance and volume distensibility of the isolated dogfish gut and segments of dogfish arteries and veins were investigated. The volume-distensibility curves for dogfish arteries and veins are very similar to comparable curves derived from arteries and veins of dogs or man. The vascular volume-distensibility curve of the gut, however, shows a greater distensibility at higher systemic pressure than at lower pressure. Evidence is presented that significant amounts of fluid leave the vascular compartment at a lower systemic pressure than in an isolated dog hindlimb preparation. However, this alone does not explain the atypical vascular volume-distensibility curve obtained from the dogfish gut. It is suggested that in the dogfish capillary filtration pore enlargement takes place at a very low capillary pressure or volume (compared to mammals) and this complicates the construction of a volume-distensibility curve because initial vascular volume is not constant.

Animals↗

Vascular response of the dogfish and sculpin to angiotensin II.

The mechanism by which angiotensin II (AII) exerts its pressor effect was examined in a saltwater teleost and elasmobranch. In vivo application of AII failed to elicit any changes in dogfish (Squalus acanthias) mesenteric microcirculatory blood flow, but the sculpin (Myoxocephalus octodecimspinosus) mesentery bed showed consistent reversible decreases in blood flow following AII application. Topical treatment with 0.5 mg phentolamine did not attenuate the AII response. In vitro addition of AII did not alter the mean resting tension of the dogfish celiac artery or intestinal vein, but significantly contracted the sculpin aorta. Addition of chromaffin tissue to the dogfish arterial muscle bath imparted significant vasoaction to AII when compared to saline. Both in vivo and in vitro experiments demonstrate the absence of functional AII vascular receptors in dogfish and their presence in the sculpin. Additional evidence for the mediation of AII vascular action in the dogfish by catecholamine release has been demonstrated.

Angiotensin II↗

Effect of ganglionic blockade on catecholamine secretion in exercised dogfish.

A brief bout of vigorous exercise results in significant increases in plasma epinephrine (E) and norepinephrine (NE) in the dogfish, Squalus acanthias. Since the presence of a functioning sympathetic nervous system in dogfish is in doubt, experiments were undertaken to show whether or not exercise-induced catecholamine (CA) secretion is under autonomic neurogenic control. Changes in plasma E and NE in a control group of exercised fish were compared with changes in fish exercised while under the influence of ganglionic blockade. Ganglionic blockade was induced in dogfish by hexamethonium infusion before exercise. CA secretion in response to a subsequent bout of exercise was significantly reduced without impairment of the ability of the fish to exercise. The pattern of systemic arterial pressure response to exercise and recovery (initial decrease during exercise followed by a prompt recovery to control level) was not significantly altered by ganglionic blockade. It is concluded that in dogfish some fraction of CA secretion capacity is possibly or potentially under neurogenically related control. Apparently the fraction of CA secretion under such control is not essential for performing exercise. The pattern of CA secretion accompanying the events of exercise and recovery in dogfish suggests that CA may play a more important role in recovery from exercise than in its performance.

Animals↗

Escape manoeuvres in the spiny dogfish (Squalus acanthias).

The locomotor performance of dogfish during escape responses was observed by means of high-speed video. Dogfish show C-type escape responses that are comparable with those shown previously in teleosts. Dogfish show high variability of turning rates of the anterior part of the body (head to centre of mass), i.e. with peak values from 434 to 1023 deg. s(-1). We suggest that this variability may be due to the presence of two types of escape manoeuvres, i.e. responses with high and low turning rates, as previously found in a teleost species. Fast responses (i.e. with high maximum turning rates, ranging between 766 and 1023 deg. s(-1)) showed significantly higher locomotor performance than slow responses (i.e. with low maximum turning rates, ranging between 434 and 593 deg. s(-1)) in terms of distance covered, speed and acceleration, although no differences were found in the turning radius of the centre of mass during the escape manoeuvres. The existence of two types of escape responses would have implications in terms of both neural control and muscular activation patterns. When compared with literature data for the locomotor performance of bony fishes, dogfish showed relatively low speed and acceleration, comparable turning rates and a turning radius that is in the low part of the range when compared with teleosts, indicating relatively high manoeuvrability. The locomotor performance observed in dogfish is consistent with their morphological characteristics: (1) low locomotor performance associated with low thrust developed by their relatively small posterior depth of section and (2) relatively high manoeuvrability associated with their high flexibility.

Acceleration↗

Plasma catecholamines in the lesser spotted dogfish and rainbow trout at rest and during different levels of exercise.

The hypothesis that there is an increase in plasma catecholamines during exercise in fish and that they play an important role in the cardiovascular adjustments during exercise was investigated in the lesser spotted dogfish and rainbow trout. In resting fish plasma catecholamines were at a concentration of 10(-9)-10(-8) mol l-1. During spontaneous swimming in the dogfish, adrenaline increased by 3.3 times to 1.9 X 10(-8) mol l-1 and noradrenaline increased by 2.3 times to 3.2 X 10(-8) mol l-1. In rainbow trout swimming at a steady 1 body length s-1 (Ls-1) in a water channel, the levels of both amines decreased to approximately 25% of the resting values. When swimming to apparent exhaustion at approximately 2 Ls-1, adrenaline was 10 times the resting value at 1.4 X 10(-8) mol l-1, whereas noradrenaline was 2.2 times the resting value at 2.3 X 10(-8) mol l-1. Only after repeated burst swimming for 2-3 min did the levels of plasma catecholamines increase substantially above the resting values. In the dogfish, both amines were at 10(-7) mol l-1, whereas in the rainbow trout, noradrenaline was at 8.5 X 10(-8) mol l-1 and adrenaline was at 2 X 10(-7) mol l-1. These levels were compared with the concentrations of catecholamines used by other workers to elicit changes in the branchial vasculature, gas exchange at the gills or gas transport to the tissues. In lesser spotted dogfish, the levels of adrenaline and noradrenaline present in the plasma during spontaneous swimming have 80% and 50% of maximum effect on gill blood vessels, respectively, whereas in rainbow trout the levels present when swimming to apparent exhaustion have approximately 20% of maximum effect on the branchial vasculature. The levels present in the trout after repeated burst swimming have 40% of maximum effect on blood vessels in the gills. The difference between the dogfish and the trout may be related to the lack of innervation of the gill blood vessels in the former. Enhancement of gas exchange across the gills of rainbow trout can be demonstrated by using adrenaline at the concentration found after repeated burst swimming. It is possible, however, that the concentration of adrenaline found in the plasma of trout after swimming to apparent exhaustion may cause an increase in the concentration of oxygen in arterial blood, thus enhancing oxygen delivery to the tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Does gill boundary layer carbonic anhydrase contribute to carbon dioxide excretion: a comparison between dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss).

In vivo experiments were conducted on spiny dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss) in sea water to determine the potential role of externally oriented or gill boundary layer carbonic anhydrase in carbon dioxide excretion. This was accomplished by assessing pH changes in expired water using a stopped-flow apparatus. In dogfish, expired water was in acid-base disequilibrium as indicated by a pronounced acidification (delta pH=-0.11+/-0.01; N=22; mean +/- s.e.m.) during the period of stopped flow; inspired water, however, was in acid-base equilibrium (delta pH=-0.002+/-0.01; N=22). The acid-base disequilibrium in expired water was abolished (delta pH=-0.005+/-0.01; N=6) by the addition of bovine carbonic anhydrase (5 mg l-1) to the external medium. Addition of the carbonic anhydrase inhibitor acetazolamide (1 mmol l-1) to the water significantly reduced the magnitude of the pH disequilibrium (from -0.133+/-0.03 to -0.063+/-0.02; N=4). However, after correcting for the increased buffering capacity of the water caused by acetazolamide, the acid-base disequilibrium during stopped flow was unaffected by this treatment (control delta [H+]=99.8+/-22.8 micromol l-1; acetazolamide delta [H+]=81.3+/-21.5 micromol l-1). In rainbow trout, expired water displayed an acid-base disequilibrium (delta pH=0.09+/-0.01; N=6) that also was abolished by the application of external carbonic anhydrase (delta pH=0.02+/-0.01). The origin of the expired water acid-base disequilibrium was investigated further in dogfish. Intravascular injection of acetazolamide (40 mg kg-1) to inhibit internal carbonic anhydrase activity non-specifically and thus CO2 excretion significantly diminished the extent of the expired water disequilibrium pH after 30 min (from -0.123+/-0.01 to -0.065+/-0.01; N=6). Selective inhibition of extracellular carbonic anhydrase activity using a low intravascular dose (1.3 mg kg-1) of the inhibitor benzolamide caused a significant reduction in the acid-base disequilibrium after 5 min (from -0.11+/-0.01 to -0.07+/-0. 01; N=14). These results demonstrate that the expired water acid-base disequilibrium originates, at least in part, from excretory CO2 and that extracellular carbonic anhydrase in dogfish may have a significant role in carbon dioxide excretion. However, externally oriented carbonic anhydrase (if present in dogfish) plays no role in catalysing the hydration of the excretory CO2 in water flowing over the gills and thus is unlikely to facilitate CO2 excretion.

Acetazolamide↗

Characterisation of C-type natriuretic peptide receptors in the gill of dogfish Triakis scyllia.

Only C-type natriuretic peptide (CNP) has been identified in primitive elasmobranch fish. CNP is the most conserved molecule in the natriuretic peptide family, suggesting that it is the ancestral type. As a first step to investigating the ancestral type of natriuretic peptide receptors, CNP receptors were characterised in an elasmobranch (dogfish, Triakis scyllia) by radioligand-binding analysis using 125I-[Tyr0]-dogfish (df)CNP. None of the modifications of the CNP molecule that occur at the time of iodination (addition of a Tyr residue at the N-terminus, introduction of iodine into Tyr0 or oxidation of Met17) affect the affinity of dfCNP for the receptors. Neither did oxidation of Met17 decrease the ability of CNP to stimulate cGMP production. In the tissues examined, CNP receptors were densest in the gill cells followed by the intestine, interrenal gland and rectal gland, all of which are involved in osmoregulation in elasmobranchs. CNP-stimulated guanylate cyclase (GC) activity was highest in the interrenal gland, intestine, brain and rectal gland, followed by the gill cells. Since the gill cells seem to contain both GC-coupled and uncoupled receptors, this tissue was used to characterise dogfish CNP receptors. Scatchard analysis of the saturation isotherm revealed two classes of binding site: one has a Kd of 24.0 pM and Bmax of 59.9 fmol/mg protein, and the other has low affinity (Kd > 1 nM) and high capacity (Bmax > 200 fmol/mg protein). The higher-affinity binding sites may represent GC-uncoupled receptors, because C-ANF, a specific ligand for GC-uncoupled receptors, almost completely displaced CNP binding. Affinity-labelling experiments showed that dogfish receptors have molecular masses of about 90, 170 and 340 kDa, and CNP binding to the former two receptors is inhibited by C-ANF. After reduction with 2-mercaptoethanol, most 170 kDa labelling was shifted to 90 kDa. It is concluded that GC-uncoupled receptors in the dogfish gill have higher molecular mass than those of mammals and eel (about 65 kDa), and are present mostly as monomers even in non-reducing conditions. However, a small population of GC-coupled receptors is also present, as demonstrated by an increase in cGMP production.

Animals↗

Isolation and characterization of two major serum proteins from the dogfish, Mustelus canis, C-reactive protein and amyloid P component.

Two major serum components from the dogfish, Mustelus canis, have been isolated using affinity chromatography. Both proteins bind to an AH-Sepharose 4B-phosphorylcholine affinity matrix in the presence of Ca2+ and are eluted from the column by EDTA. Upon readdition of Ca2+ to the eluted proteins, the two proteins can be separated by passage through a column of Sepharose CL-4B. The first protein, C-reactive protein, passes through the Sepharose CL-4B column in the presence of Ca2+ whereas the second protein, serum amyloid P component, remains bound. The serum amyloid P component is then eluted from the Sepharose CL-4B in pure form by EDTA. The dogfish C-reactive protein isolated by the phosphorylcholine affinity matrix precipitates with pneumococcal C-polysaccharide and with a synthetic derivative of bovine serum albumin to which phosphorylcholine is covalently attached. The precipitation is inhibited by either EDTA or by phosphorylcholine. Dogfish C-reactive protein has a molecular weight of approximately 250,000 with dimeric subunits of Mr = 50,000. Upon addition of beta-mercaptoethanol these dimeric subunits dissociate to two identical monomeric subunits of Mr = 25,000. The protein cross-reacts immunologically with goat antisera prepared against rabbit C-reactive protein. The dogfish serum amyloid P component has a molecular weight of at least 250,000 with monomeric subunits of Mr = 25,000. Cross-reactivity of the amyloid P component with the C-reactive protein could not be shown. However, NH2-terminal sequence analysis of the first 20 amino acids showed some homology. The relationship of dogfish C-reactive protein to the C-reactive proteins in Limulus polyphemus and in rabbits and humans is discussed.

Amino Acid Sequence↗