Gastrin/CCK-like peptides in the spiny dogfish, Squalus acanthias; concentrations and actions in the gut

1. The presence of gastrin/CCK-like immunoreactive material in both muscle and mucosal layers of the whole gut, except the cardiac stomach, in Squalus acanthias has been confirmed by radioimmunoassay. The highest levels were measured in rectum and spiral intestine respectively. 2. Fractionation of the spiral intestine mucosal extract on DEAE 52 columns indicated the presence of multiple forms of gastrin/CCK in the elasmobranch gut. 3. Synthetic mammalian gastrin- and CCK-like peptides, when effective, increased the rhythmic activity and sometimes the basal tonus in smooth muscle preparations from the intestine or rectum. 4. The irregular effects of mammalian peptides on the motility indicated an essential difference from the mammalian counterpart in the structure of the elasmobranch peptide present in the gastrointestinal nerves.     

The effect of reduced salinity on tissue and plasma composition of the dogfish,Squalus acanthias

• 1.1. Dogfish (Squalus acanthias) were acclimated to reduced salinities and their plasma, muscle tissue and erythrocytes subsequently analysed.
• 2.2. Decrease in the osmolarity of the plasma was principally due to a fall in urea concentration and a significant fall in the concentrations of sodium and chloride.
• 3.3. Changes in the muscle and erythrocytes in dilute media were a decrease in urea, potassium, sodium and chloride concentrations.
• 4.4. The concentrations of the free amino acids in the muscle and the red blood cells decreased more than would be expected by the movements of water only.
• 5.5. The results were discussed in relation to the regulation of cellular volume and the involvement of the free amino acid pool of the tissues in this process.

     

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.     

The primary structure of the hemoglobin of the dogfish shark (Squalus acanthias). Antagonistic effects of ATP and urea on oxygen affinity of an elasmobranch hemoglobin

The amino-acid sequence of the hemoglobin of the Dogfish Shark (Squalus acanthias) is presented. The alpha-chains consist of 141 residues and show a Thr/Ser ambiguity at position 3. The beta-chains consist of 142 residues and evidently have no D-helix; they show an Asn/Tyr ambiguity at position 104. Both chains have free N-terminal amino acids. The phylogenetic distance from the human alpha- and beta-chains is indicated by 49.3% and 56.2% amino-acid exchanges. The primary structure is discussed in relation to the oxygen-binding properties of elasmobranch hemoglobin, particularly as regards the antagonistic effects of urea and ATP, and the effects of proton concentration (the alkaline and acid Bohr effects, and the Root effect).     

Activation of liver carnitine palmitoyltransferase-1 and mitochondrial acetoacetyl-CoA thiolase is associated with elevated ketone body levels in the elasmobranch Squalus acanthias

Elasmobranch fishes are an ancient group of vertebrates that have unusual lipid metabolism whereby storage lipids are mobilized from the liver for peripheral oxidation largely as ketone bodies rather than as nonesterified fatty acids under normal conditions. This reliance on ketones, even when feeding, implies that elasmobranchs are chronically ketogenic. Compared to specimens sampled within 2 d of capture (recently captured), spiny dogfish Squalus acanthias that were held for 16-33 d without apparent feeding displayed a 4.5-fold increase in plasma concentration of d- beta -hydroxybutyrate (from 0.71 to 3.2 mM) and were considered ketotic. Overt activity of carnitine palmitoyltransferase-1 in liver mitochondria from ketotic dogfish was characterized by an increased apparent maximal activity, a trend of increasing affinity (reduced apparent K(m); P=0.09) for l-carnitine, and desensitization to the inhibitor malonyl-CoA relative to recently captured animals. Acetoacetyl-CoA thiolase (ACoAT) activity in isolated liver mitochondria was also markedly increased in the ketotic dogfish compared to recently captured fish, whereas no difference in 3-hydroxy-3-methylglutaryl-CoA synthase activity was found between these groups, suggesting that ACoAT plays a more important role in the activation of ketogenesis in spiny dogfish than in mammals and birds.     

Central venous pressure and mean circulatory filling pressure in the dogfish Squalus acanthias: adrenergic control and role of the pericardium

Subambient central venous pressure (Pven) and modulation of venous return through cardiac suction (vis a fronte) characterizes the venous circulation in sharks. Venous capacitance was estimated in the dogfish Squalus acanthias by measuring the mean circulatory filling pressure (MCFP) during transient occlusion of cardiac outflow. We tested the hypothesis that venous return and cardiac preload can be altered additionally through adrenergic changes of venous capacitance. The experiments involved the surgical opening of the pericardium to place a perivascular occluder around the conus arteriosus. Another control group was identically instrumented, but lacked the occluder, and was subjected to the same pharmacological protocol to evaluate how pericardioectomy affected cardiovascular status. Routine Pven was negative (-0.08+/-0.02 kPa) in control fish but positive (0.09+/-0.01 kPa) in the pericardioectomized group. Injections of 5 microg/kg body mass (Mb) of epinephrine and phenylephrine (100 microg/kg Mb) increased Pven and MCFP, whereas isoproterenol (1 microg/kg Mb) decreased both variables. Thus, constriction and relaxation of the venous vasculature were mediated through the respective stimulation of alpha- and beta-adrenergic receptors. Alpha-adrenergic blockade with prazosin (1 mg/kg Mb) attenuated the responses to phenylephrine and decreased resting Pven in pericardioectomized animals. Our results provide convincing evidence for adrenergic control of the venous vasculature in elasmobranchs, although the pericardium is clearly an important component in the modulation of venous function. Thus active changes in venous capacitance have previously been underestimated as an important means of modulating venous return and cardiac performance in this group.     

Bombesin-like immunoreactivity in skates and the in vitro effect of bombesin on coronary vessels from the longnose skate, Raja rhina.

Bombesin-like immunoreactivity is present in nerve fibers projecting to the cardiovascular system, including the coronary arteries, and to the gastrointestinal canal, and in endocrine cells of the gut of skates belonging to the family Rajidae. Synthetic bombesin contracted isolated coronary rings from the longnose skate, Raja rhina, in a cumulative fashion. The contractile response was 84% of that of 60 mM potassium chloride. The pD2-value for bombesin was 8.83 (S.E.M. = 0.33; n = 15). Phentolamine, atropine and two substance P-antagonists increased the sensitivity to bombesin, while atenolol, sotalol, nifedipine, tetrodotoxin and two bombesin antagonists were devoid of significant effects. We conclude from this study that a bombesin-like peptide is present in nerves innervating the cardiovascular system and the gastrointestinal canal of skates of the family Rajidae, and that bombesin contracts coronary vessels in vitro via a direct mechanism and/or via mechanisms involving alpha-adrenergic and muscarinergic receptors.     

The effect of mercury on chloride secretion in the shark (Squalus acanthias) rectal gland

1. Mercuric chloride inhibited chloride secretion in a dose dependent way in isolated perfused rectal glands. The effect was readily apparent at a concentration of 10⁻⁶ M and profound and irreversible at 10⁻⁴ M.2. The dithiol dithiothreitol (DTT) 10⁻² M completely prevented the effect of 10⁻⁶ M mercuric chloride, reduced that at 10⁻⁵M and 10⁻⁴M, and made the inhibition at the latter concentration reversible.3. Two organic mercurials, mersalyl and meralluride, that are effective diuretics in the mammalian kidney, and p-chloromercuribenzoyl sulfonic acid (PCMBS), that has no diuretic activity, had no effect on chloride secretion by the rectal gland.4. The effect of mersalyl was not modified by lowering the pH of the solution perfusing the glands.5. These results indicate that inorganic mercury and organic mercurials do not share the same mechanism of action.6. The absence of an effect of organic mercurials on chloride transport in the rectal gland suggests that its effect on another chloride transporting epithelia, the thick ascending limb of the loop of Henle, is not mediated by inhibition of the chloride cotransporter or Na⁺, K⁺-ATPase, common to both epithelia.     

The role of the pericardium in cardiac function in the dogfish, Squalus acanthias

Opening the pericardium to the ambient bathing fluid surrounding the in situ perfused dogfish ( Squalus acanthias ) heart caused a precipitous fall in cardiac output. Cardiac output fell by 55% despite the rise of mean input pressure from subambient, to near zero levels. Lower cardiac output caused a fall in mean output pressure but not diastolic pressure as this was set by the experimenters. With the pericardium intact, the heart was filled by suction. With an open pericardium the magnitude of negative input pressures was severely reduced. None the less, far short periods within the cardiac cycle, the heart was still able to generate subambient pressures in the atrium and so draw fluid from the central veins.     

Vasoactive receptors in abdominal blood vessels of the dogfish shark, Squalus acanthias

Previous studies have demonstrated that the ventral aorta of the dogfish shark, Squalus acanthias, responds to a variety of cell-signaling agents. To investigate the generality of vasoactive receptors in the shark vasculature, in particular a conductance artery (anterior mesenteric) and vein (posterior intestinal), I measured the effect of acetylcholine, endothelin, nitric oxide, natriuretic peptides, and prostaglandins on tension in isolated rings from these vessels. Both vessels responded to these agents, and responses to receptor-specific ligands for endothelin and natriuretic peptide receptors suggest that B-type endothelin receptors are expressed in both vessels and that the artery expresses both A- and B-type natriuretic peptide receptors; however, the vein (like the ventral aorta) expresses only the B-type natriuretic peptide receptor. My data suggest that a suite of signaling systems is ubiquitous in both arteries and veins in at least this elasmobranch species. Their role in hemodynamics and osmoregulation (perfusion of gill and rectal gland) remains to be determined.     

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.