Bilirubin metabolism in the spiny dogfish, Squalus acanthias, and the small skate, Raja erinacea.

1. The main bilirubin conjugate in bile of spiny dogfish (Squalus Acanthias) and small skate (Raja Erinacea) is bilirubin monoglucuronide. 2. Microsomal preparations from dogfish and small skate liver have similar bilirubin UDPglucuronyltransferase (UDPGT) activity and catalyze the conjugation of bilirubin with glucose from UDPglucose. 3. The activity of bilirubin glucosidation (UDPGT) was 0.5 times UDPG1T activity in dogfish and 0.15 times in skate liver microsomes. 4. Sodium cholate increased UDPGT and UDPG1T activities in dogfish and skate liver microsomal preparations only minimally, but the detergent markedly increased thermolability of UDPGT in skate liver microsomes.     

Submitochondrial localization of arginase and other enzymes associated with urea synthesis and nitrogen metabolism, in liver of Squalus acanthias

The submitochondrial localization of the four mitochondrial enzymes associated with urea synthesis in liver of Squalus acanthias (spiny dogfish), a representative elasmobranch, was determined. Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, ornithine carbamoyltransferase, glutamine synthetase, and arginase were all localized within the matrix of liver mitochondria. The subcellular and submitochondrial localization and activities of several related enzymes involved in nitrogen metabolism and gluconeogenesis in liver and dogfish are also reported. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were localized in the mitochondrial matrix. Synthesis of citrulline by isolated mitochondria from ornithine proceeds at a near optimal rate at ornithine concentrations as low as 0.08 mM. The same stoichiometry and rates of citrulline synthesis are observed when ornithine is replaced by arginine. The mitochondrial location of arginase does not appear to reflect a mechanism for regulating ornithine availability.     

Bombesin-like immunoreactive material in the gut, and the effect of bombesin on the stomach circulatory system of an elasmobranch fish, Squalus acanthias

The distribution, nature and amount of bombesin-like immunoreactivity (IR) in the gastrointestinal canal and its afferent vessels was investigated in the spiny dogfish (Squalus acanthias) together with the in vitro effect of synthetic bombesin on perfusion flow through the vascularly perfused dogfish stomach. Nerve fibres showing bombesin-like IR frequently occurred in the walls of the anterior mesenteric and coeliac arteries and the intrinsic vessels of the gut. Chromatographic studies revealed that multiple peaks of bombesin-like IR material were present in extracts of the spiny dogfish gastrointestinal vessels. Bombesin-like IR was also present in muscle and mucosal layers of the gut with higher levels in muscle compared with mucosa, and higher levels in the stomach than in the intestine and the rectum. Exogenous bombesin increased the flow through the vasculary perfused spiny dogfish stomach in a dose-dependent manner. Studies with tetrodotoxin and atropine showed that bombesin probably exerts its effect directly on the vascular musculature. It is concluded from this study that bombesin-like material is present in nerves innervating the gut circulatory system of the spiny dogfish. Bombesin may affect the blood-flow to the gastrointestinal canal, possibly via a direct effect on vascular smooth muscle.     

Purification and properties of mitochondrial phosphoenolpyruvate carboxykinase from liver of Squalus acanthias

Liver from Squalus acanthias (spiny dogfish), a representative elasmobranch, contains approximately 1.4 units (mumol/min) of phosphoenolpyruvate carboxykinase activity per gram and approximately 90% of the total units of activity are localized in the mitochondria. The mitochondrial phosphoenolpyruvate carboxykinase was isolated and characterized. The purified enzyme has properties generally similar to those found in mammalian and avian species. The enzyme has a molecular weight of approximately 70,000 and exists in a functional state as a monomer. The isolated enzyme displays a dual cation requirement (e.g., 6 mM Mg2+ and 10 microM Mn2+) for maximal activity; very little activity is observed when Mg2+ is present alone, and the maximal activity attained with Mn2+ alone (millimolar concentrations required) is significantly less than that observed under optimal conditions with both cations present. When assayed in the direction of oxalacetate formation there is a lag in product formation with time; the lag can be eliminated by the presence of 50 microM GTP (product). The Km for substrates is not affected by Mn2+ concentration, suggesting that the role of Mn2+ may not be related to substrate binding. The apparent Km for phosphoenolpyruvate (approximately 1 mM) is substantially higher than that reported for phosphoenolpyruvate carboxykinase from other species. The activity of phosphoenolpyruvate carboxykinase is increased 70% by physiological concentrations of urea. Maximal velocity of the reaction in the direction of oxalacetate formation is approximately half that of the reverse reaction.     

14C]palmitate uptake in isolated rat liver mitochondria: effects of fasting, diabetes mellitus, and inhibitors of carnitine acyltransferase.

The rapid association of Na-[16-(14)C]palmitate with isolated rat liver mitochondria was measured by an oil separation method. This association was time and temperature-dependent and was absolutely dependent on the presence of exogenous ATP and CoASH and partially dependent on exogenous carnitine. Carnitine dependence was enhanced at lower concentrations of [(14)C]palmitate. At 6.5 micro M [(14)C]palmitate (molar ratio of palmitate to albumin equal to 0.54), the rate of association was linear for 20 sec and was increased more than 100% in the presence of carnitine. Carnitine-dependent association was inhibited by 2-bromopalmitate, an inhibitor of carnitine acyltransferase I, but not by (+)-octanoylcarnitine, a presumed inhibitor of carnitine acyltransferase II. The association of [(14)C]palmitate with mitochondria was enhanced from 190 to 330% in mitochondria isolated from fasted animals and from 160 to 230% in mitochondria isolated from diabetic, ketotic animals as compared to control animals. The enhanced association with mitochondria from fasted animals was inhibited by 2-bromopalmitate. These studies demonstrate a method of evaluating fatty acid association with mitochondria which, because of its dependence on carnitine and carnitine acyltransferase I activity, most likely represents true uptake into mitochondria. Furthermore, these studies indicate that the carnitine-dependent uptake of fatty acids into mitochondria is enhanced in the two ketotic states evaluated and that the carnitine acyltransferase system may be a regulatory site in ketone body production.     

Global population structure of the spiny dogfish Squalus acanthias,a temperate shark with an antitropical distribution

The spiny dogfish (Squalus acanthias) is a temperate, coastal squaloid shark with an antitropical distribution in the Atlantic and Pacific oceans. The global population structure of this species is poorly understood, although individuals are known to undergo extensive migrations within coastal waters and across ocean basins. In this study, an analysis of the global population structure of the spiny dogfish was conducted using eight polymorphic nuclear microsatellite markers and a 566‐bp fragment of the mitochondrial ND2 gene region. A low level of genetic divergence was found among collections from the Atlantic and South Pacific basins, whereas a high level of genetic divergence was found among Pacific Ocean collections. Two genetically distinct groups were recovered by both marker classes: one exclusive to North Pacific collections, and one including collections from the South Pacific and Atlantic locations. The strong genetic break across the equatorial Pacific coincides with major regional differences in the life‐history characters of spiny dogfish, suggesting that spiny dogfish in areas on either side of the Pacific equator have been evolving independently for a considerable time. Phylogeographic analyses indicate that spiny dogfish populations had a Pacific origin, and that the North Atlantic was colonized as a result of a recent range expansion from the South American coast. Finally, the available data strongly argue for the taxonomic separation of the North Pacific spiny dogfish from S. acanthias and a re‐evaluation of the specific status of S. acanthias is warranted.     

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.     

A comparison of dogfish and bovine chymotrypsins

Bovine and dogfish chymotrypsins were compared to determine if chymotrypsin from a poikilothermic organism (spiny dogfish (Squalus acanthias] adapted to low temperatures possessed catalytic properties different from those of the same enzyme from a warm-blooded animal. An improved procedure was developed for isolating dogfish pancreatic chymotrypsin. The least hydrophobic and smallest substrate used, p-nitrophenyl acetate, had similar enthalpies of association (delta Ha) with both enzymes, whereas larger, more hydrophobic substrates had delta Ha values that were of opposite sign for the two enzymes. As the temperature increased, the association constants (1/Ks) for p-nitrophenyl valerate and p-nitrophenyltrimethyl acetate increased for dogfish chymotrypsin and decreased for bovine chymotrypsin, while the free energies of association (delta Ga) remained relatively constant. Acylation of chymotrypsin was 1.5-2.5 times slower in the dogfish enzyme than in the bovine enzyme except below 15 degrees C with p-nitrophenyltrimethyl acetate. delta H++ for acylation by p-nitrophenyltrimethyl acetate were 2.0 kcal/mol for the dogfish enzyme and 10.2 kcal/mol for the bovine, whereas delta H++ values were only slightly lower in the dogfish enzyme for the other two substrates. For all substrates, the deacylation rate constant (kcat) was greater with dogfish chymotrypsin than bovine. However, the free energies of activation (delta G++) for deacylation were nearly equal between the two enzymes for each of the substrates.     

Further evidence for ancient role of ACTH peptides at melanocortin (MC) receptors; pharmacology of dogfish and lamprey peptides at dogfish MC receptors

The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand-receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey (Petromyzon marinus) and spiny dogfish (Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish alpha-, beta- and gamma-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than alpha-MSH to the dogfish MC4 receptor. We also show that dogfish delta-MSH is able to bind to MC receptors and its potency is higher than of dogfish beta-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.     

Diet of spiny dogfish, Squalus acanthias Linnaeus, on the east coast, South Island, New Zealand

Stomach content data from 7283 spiny dogfish, Squalus ucunthius Linnaeus, from east coast South Island of New Zealand are presented. The major dietary components based on occurrence were 60% crustaceans and 15% fish. The principal food items were the post-larval phase of the squat lobster, Munida greguria Fabricius (30%), and the euphausiid, Nyctiphanes australis Sars (20%). Commercial fish species contributed to less than 5% of the diet. Diet of dogfish varied with dogfish size and sex. Compared to northern hemisphere studies, spiny dogfish in this study area had (1) a high proportion of crustaceans in their diet, (2) a relatively low percentage of empty stomachs, (3) a relatively high incidence of cannibalism.     

The influence of anthropogenic pollution on several indicators of congenital immunity and oxidizing processes in the liver of Squalus acanthias Linnaeus, 1758 (Pisces: Squalidae) that inhabit coastal waters of the Black Sea

The results of a study on several parameters of congenital immunity and oxidative processes in the liver of spiny dogfish Squalus acanthias that inhabit the Caucasus coastal waters of the Black Sea with various anthropogenic loads are described. The antimicrobial properties of serum, the content of non-specific immune complexes in the liver, the total lipid value, and antioxidant activity were studied. It was determined that the ratio of immunodeficient individuals was much higher in samples from the polluted areas than in samples from the clean ones. The fish sampled in areas of high anthropogenic load contained higher contents of products of lipid peroxidation and immune complexes, as well as lower values of antioxidants.     

A comparative study of the transport of pyruvate in liver mitochondria from normal and diabetic rats

A comparative study of the transport of pyruvate in liver mitochondria from normal and diabetic rats has been carried out. TheK _m for net pyruvate uptake in diabetic, ketotic mitochondria is practically equal to that measured in normal mitochondria, while theV _max is significantly lower. The lower activity of the pyruvate translocator in diabetic mitochondria compared to normal mitochondria is also shown by swelling experiments as well as by following the rate of pyruvate-supported respiration. Pre-exposure of mitochondria from normal rats to the ketone body acetoacetate and to 2-oxobutyrate results in a decrease of theK _m for pyruvate uptake. This effect is impaired in mitochondria from diabetic animals. The results indicate that the activity and the properties of the mitochondrial pyruvate translocator are modified in the diabetic, ketotic condition.Supported by a joint grant from Consiglio Nazionale delle Ricerche, Rome, Italy, and the Polish Academy of Sciences, Warsaw, Poland.     

Do abiotic and ontogenetic factors influence the diet of a generalist predator? Feeding ecology of the Pacific spiny dogfish (<Emphasis Type="Italic">Squalus suckleyi</Emphasis>) in the northeast Pacific Ocean

Stomach contents from 1221 Pacific spiny dogfish (Squalus suckleyi) were collected from the Gulf of Alaska over 2004–2006 and analyzed to determine predominant prey species. Pacific spiny dogfish in the Gulf of Alaska have a variable diet suggesting that they are generalist feeders. Of the 68.30% of stomachs with prey contents, the most important prey groups based on the percent prey-specific index of relative importance (%PSIRI) were shrimp (27.06%), cephalopods (17.16%), and forage fish (17.11%). Remaining components of the diet were inconsistent. Commercially valuable species, such as salmon, Oncorhynchus spp., and rockfish, Sebastes spp. constituted only 2.37% and 1.51% of the diet of Pacific spiny dogfish, respectively. Diet diversity was not significantly influenced by spatial, temporal or ontogenetic factors; however, trophic level of prey and average prey item weight varied both interannually and with ontogeny. With increasing size, Pacific spiny dogfish incorporate larger prey items into their diet, and diet composition may be driven more by seasonal availability and prey size than any other factors.     

Characterization and isolation of DNA microsatellite primers in the spiny dogfish (Squalus acanthias)

The North Atlantic spiny dogfish (Squalus acanthias) population has been declining since the 1980s. Proper management of this population is essential as dogfish are prone to rapid collapse based on the unique biology of this species. We characterized eight microsatellite loci for spiny dogfish to use in determining the genetic structure of this species along the Atlantic coast of the USA.     

Localization of ouabain-sensitive,potassium-dependent nitrophenyl phosphatase in the rectal gland of the spiny dogfish,Squalus acanthias

Tissue from the digitiform rectal gland of the spiny dogfish, Squalus acanthias, was fixed briefly by formaldehyde perfusion and studied for the specificity and localization of p-nitrophenyl phosphatase (NPP'ase) activity. The enzymatic activity was K+-dependent (56%) and ouabain-sensitive (67%-inhibition). The electron-dense reaction product (SrPO4) of the cytochemical reaction (Ernst, 1972b) was localized along the inner surfaces of the basolateral membranes of the secretory cells. It was absent from mitochondria nuclei, vesicles, and other organelles. The luminal surface of the secretory cells was slightly reactive. On the basis of (1) this pattern of localization for the sodium transport system, (2) the presence of extensive intercellular labyrinthine channels (Bulger, 1963) that would facilitate "standing gradients" (Diamond and Bossert, 1968), and (3) the specific distribution of the energy-providing mitochondria, we conclude that the concentration and electrochemical gradients recorded from the secreting gland (Hayslett et al., 1974) are maintained across the domains of the basolateral surfaces of the secretory cells.     

Some kinetic properties of dogfish chymotrypsin

A comparison of some kinetic properties was made between bovine chymotrypsin and chymotrypsin isolated from the spiny dogfish (Squalus acanthias). The major difference between the two enzymes was observed in the molecular activity (kcat), with the dogfish enzyme being two to three times more active than the bovine enzyme. The exact difference was dependent on the substrate and assay conditions. The two enzymes showed similar kinetic properties with respect to the following: similar inhibition by indole and naphthol derivatives, activities vs BTEE and a series of n-fatty acid esters of p-nitrophenol, KM values, optimal pH and temperature and activation energies.     

Reply to Hussey et al.: The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks

Carbon and nitrogen stable isotope analyses have improved our understanding of food webs and movement patterns of aquatic organisms. These techniques have recently been applied to diet studies of elasmobranch fishes, but isotope turnover rates and isotope diet–tissue discrimination are still poorly understood for this group. We performed a diet switch experiment on captive sandbar sharks (Carcharhinus plumbeus) as a model shark species to determine tissue turnover rates for liver, whole blood, and white muscle. In a second experiment, we subjected captive coastal skates (Leucoraja spp.) to serial salinity reductions to measure possible impacts of tissue urea content on nitrogen stable isotope values. We extracted urea from spiny dogfish (Squalus acanthias) white muscle to test for effects on nitrogen stable isotopes. Isotope turnover was slow for shark tissues and similar to previously published estimates for stingrays and teleost fishes with low growth rates. Muscle isotope data would likely fail to capture seasonal migrations or diet switches in sharks, while liver and whole blood would more closely reflect shorter term movement or shifts in diet. Nitrogen stable isotope values of skate blood and skate and dogfish white muscle were not affected by tissue urea content, suggesting that available diet–tissue discrimination estimates for teleost fishes with similar physiologies would provide accurate estimates for elasmobranchs.     

Age and length at maturity of the female spiny dogfish,Squalus acanthias,in the Strait of Georgia,British Columbia,Canada

Synopsis A total of 3068 female spiny dogfish were examined to determine the age and length at maturity. The median age at maturity for females was 35.5 years with 95% confidence limits between 35.0 and 35.9 years. It was found that slower growing dogfish tended to mature at a smaller size. A deterministic model incorporating fecundity, growth and reproduction was used to examine the reproductive style of spiny dogfish. The age at maturity reported corresponds to the level that theoretically maximizes lifetime reproductive output for a cohort.     

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.