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-1-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 ~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 (~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 ~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.     

Urea and water permeability in dogfish (Squalus acanthias) gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.     

The effects of salt-induced hypertension on alpha1-adrenoreceptor expression and cardiovascular physiology in the rainbow trout (Oncorhynchus mykiss)

Experiments were conducted on rainbow trout to determine the impact of dietary salt on arterial blood pressure. After 4-6 wk, fish fed a salt-enriched diet exhibited a 37% elevation of dorsal aortic pressure (from 23.8 +/- 1.2 to 32.6 +/- 1.4 mmHg) and an 18% increase in ventral aortic pressure (from 33.0 +/- 1.5 to 38.9 +/- 1.3 mmHg). The hypertension presumably reflected the increase in cardiac output (from 31.0 +/- 0.8 to 36.4 +/- 2.2 ml.min(-1).kg(-1)) because systemic and branchial resistances were statistically unaltered by salt feeding. The chronic hypertension was associated with a decrease in the pressor responses of the systemic vasculature to catecholamines and hypercapnia in the salt-fed fish. The reduction in alpha-adrenergic responsiveness of the systemic vasculature is consistent with desensitization or loss of functional alpha-adrenoceptors (alpha-ARs). In support of this idea, the salt-fed fish exhibited significantly decreased levels of alpha(1D)-AR mRNA in the dorsal aorta and the afferent (ABA) and efferent branchial arteries (EBA). In contrast, however, the results obtained from norepinephrine dose-response curves for EBA and ABA vascular rings in vitro did not provide evidence for loss of function of branchial artery alpha(1)-ARs in the salt-fed fish. Indeed, the EC(50) for the EBA norepinephrine dose-response curve was significantly reduced (from 3.75 x 10(-7) to 2.12 x 10(-7) M) in the salt-fed fish, indicating an increase in the binding affinity of the alpha(1)-ARs.     

The influence of feeding and fasting on plasma metabolites in the dogfish shark (Squalus acanthias)

Dogfish sharks are opportunistic predators, eating large meals at irregular intervals. Here we present a synthesis of data from several previous studies on responses in plasma metabolites after natural feeding and during prolonged fasting (up to 56 days), together with new data on changes in plasma concentrations of amino acids and non-esterified fatty acids. Post-prandial and long-term fasting responses were compared to control sharks fasted for 7 days, a typical inter-meal interval. A feeding frenzy was created in which dogfish were allowed to feed naturally on dead teleosts at two consumed ration levels, 2.6% and 5.5% of body weight. Most responses were more pronounced at the higher ration level. These included increases in urea and TMAO concentrations at 20 h, followed by stability through to 56 days of fasting. Ammonia levels were low and exhibited little short-term response to feeding, but declined to very low values during the extended fast. Glucose and β-hydroxybutyrate both fell after feeding, the latter to a greater and more prolonged extent (up to 60 h), whereas acetoacetate did not change. During prolonged fasting, glucose concentrations were well regulated, but β-hydroxybutyrate increased to 2–3-fold control levels. Total plasma amino acid concentrations increased in a biphasic fashion, with peaks at 6–20 h, and 48–60 h after the meal, followed by homeostasis during the extended fast. Essential and non-essential amino acids generally followed this same pattern, though some exhibited different trends after feeding: taurine, β-alanine, and glycine (decreases or stability), alanine and glutamine (modest prolonged increases), and threonine, serine, asparagine, and valine (much larger short-term increases). Plasma non-esterified fatty acid concentrations declined markedly through 48 h after the 2.6% meal. These data are interpreted in light of companion studies showing elevations in aerobic metabolic rate, urea production, rectal gland function, metabolic base excretion, and activation of ornithine–urea cycle and aerobic enzymes after the meal, and muscle N-depletion but maintenance of osmolality and urea production during long-term fasting.     

Bomb dating and age validation using the spines of spiny dogfish (Squalus acanthias)

Bomb radiocarbon has previously been used to validate the age of large pelagic sharks based on incorporation into vertebrae. However, not all sharks produce interpretable vertebral growth bands. Here we report the first application of bomb radiocarbon as an age validation method based on date-specific incorporation into spine enamel. Our results indicate that the dorsal spines of spiny dogfish, Squalus acanthias, recorded and preserved a bomb radiocarbon pulse in growth bands formed during the 1960s with a timing which was very similar to that of marine carbonates. Using radiocarbon assays of spine growth bands known to have formed in the 1960s and 1970s as a dated marker, we confirm the validity of spine enamel growth band counts as accurate annual age indicators to an age of at least 45 year. Radiocarbon incorporation into northeast Atlantic dogfish spines occurred in similar years as those in the northwest Atlantic and northeast Pacific, although the amount of radiocarbon differed in keeping with the radiocarbon content of the different water masses. Published reports suggesting that Pacific dogfish are longer lived and slower growing than their Atlantic counterparts appear to be correct, and are not due to errors in interpreting the spine growth bands. Radiocarbon assays of fin spine enamel appears to be well suited to the age validation of sharks with fin spines which inhabit the upper 200 m of the ocean.     

Immunostimulant effects of Cotinus coggyria on rainbow trout (Oncorhynchus mykiss)

In this study, non-specific immune effects of tetra (Cotinus coggyria) on rainbow trout (Oncorhynchus mykiss) by dietary intake were investigated. Fish were fed daily ad libitum with diets containing 0.5% and 1.0% tetra for 3 weeks. After this period, fish were switched back to the basal diet for additional 6 weeks. Extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes, lysozyme activities, and total plasma protein levels were evaluated at the end of the tetra feeding period and every 3 weeks during the basal diet period. Extracellular and intracellular respiratory burst activities, phagocytic activity, lysozyme activity and total protein level parameters of the groups containing 0.5% and 1.0% tetra were higher than the control group at the end of the 3rd, 6th and 9th weeks, respectively (P < 0.05). The highest values of the non-specific immune parameters were observed in the group fed with 1.0% tetra. Tetra groups did not show any significant difference (P > 0.05) in terms of specific growth rate and average weight of the fish.     

Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.     

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.     

The modulatory effects of noradrenaline on vagal control of heart rate in the dogfish,Squalus acanthias

The possible interactions between inhibitory vagal control of the heart and circulating levels of catecholamines in dogfish (Squalus acanthias) were studied using an in situ preparation of the heart, which retained intact its innervation from centrally cut vagus nerves. The response to peripheral vagal stimulation typically consisted of an initial cardiac arrest, followed by an escape beat, leading to renewed beating at a mean heart rate lower than the prestimulation rate (partial recovery). Cessation of vagal stimulation led to a transient increase in heart rate, above the prestimulation rate. This whole response was completely abolished by 10(-4) M atropine (a muscarinic cholinergic antagonist). The degree of vagal inhibition was evaluated in terms of both the initial, maximal cardiac interval and the mean heart rate during partial recovery, both expressed as a percentage of the prestimulation heart rate. The mean prestimulation heart rate of this preparation (36+/-4 beats min(-1)) was not affected by noradrenaline but was significantly reduced by 10(-4) M nadolol (a beta-adrenergic receptor antagonist), suggesting the existence of a resting adrenergic tone arising from endogenous catecholamines. The degree of vagal inhibition of heart rate varied with the rate of stimulation and was increased by the presence of 10(-8) M noradrenaline (the normal in vivo level in routinely active fish), while 10(-7) M noradrenaline (the in vivo level measured in disturbed or deeply hypoxic fish) reduced the cardiac response to vagal stimulation. In the presence of 10(-7) M noradrenaline, 10(-4) M nadolol further reduced the vagal response, while 10(-4) M nadolol + 10(-4) M phentolamine had no effect, indicating a complex interaction between adrenoreceptors, possibly involving presynaptic modulation of vagal inhibition.     

Reproductive endocrinology of female elasmobranchs: lessons from the little skate (Raja erinacea) and spiny dogfish (Squalus acanthias).

Conventional classification of reproductive modes in female elasmobranchs fails to account for the diversity in ovarian dynamics that operate during oviparous and viviparous cycles. Delineating this diversity is crucial for understanding the endocrine regulation of the manifold physiological mechanisms utilized to retain and protect eggs and developing embryos, to fuel embryogenesis, and to manage the intrauterine milieu. Oocyte development and follicular steroidogenesis overlap with egg retention and pregnancy in some species, whereas in others the follicular phase of the cycle is temporally separated from the gravid period. A luteal phase predominates the post-ovulatory period in viviparous species. In oviparous species, the luteal phase overlaps with the follicular cycle. This heterogeneity in ovulatory cycles suggests that the endocrine system evolved a transmutable system for regulating steroidogenesis and the control of the reproductive events. The reproductive biology and endocrinology of the oviparous little skate and lecithotrophic viviparous spiny dogfish are reviewed in order to derive a working hypothesis that explains the complex nature of endocrine patterns observed in species utilizing disparate reproductive modes. An understanding of the adaptations in ovarian dynamics to particular ovulatory cycles is key to developing theories about the evolution of reproductive strategies in female elasmobranchs. J. Exp. Zool. 284:557-574, 1999.     

Isolation, purification, and the sequence of relaxin from spiny dogfish (Squalus acanthias)

A relaxin-like molecule has been isolated from the ovaries of the spiny dogfish (Squalus acanthias) which consists, like porcine relaxin, of two chains linked by the insulin-type disulfide bonds. The total number of amino acids is 54 of which 24 are in the A chain and 30 in the B chain. The molecular masses, calculated from the amino acid compositions, are 2510 Da for the A chain and 3370 Da for the B chain, making a total of 5880 Da. The N-terminus of the B chain is protected by a 5-oxoproline (pyrrolidone carboxylic acid) residue which is also found in the same position in the relaxins of sand tiger shark, pig, and man, whereas the relaxin of the rat has its 5-oxoproline residue at the N-terminal of the A chain. By all available criteria, S. acanthias relaxin is a typical member of the relaxin family although the sequence homology to mammalian relaxins is limited to about 45% of its amino acid residues. In contrast, the dogfish relaxin shows about 80% homology with sand tiger shark relaxin (the first such interspecies similarity to be observed) and has about twice the biological activity (mouse pubic symphysis test) when compared to sand tiger relaxin.     

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).     

Patterns of transgene inheritance in rainbow trout (Oncorhynchus mykiss).

There have been very few studies of the inheritance of introduced genes (transgenes) in fish. We have followed the inheritance of the mammalian fusion gene MTrGH from founder generation transgenics (originating from eggs microinjected with the MTrGH DNA) to offspring in crosses with control fish. Initial screening of the founder generation transgenics was by analysing DNA from blood samples. Only three out of six fish which carried the novel gene in blood DNA transmitted it to their offspring, despite the presence of the gene in DNA extracted from the sperm of all four male fish in this group. The frequency of transgenics in the progeny groups from the three fish which transmitted the gene varied widely: in one of these groups more than one type of MTrGH restriction pattern was found. These results suggest widespread mosaicism in founder generation transgenics.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

Variability in swimming performance and underlying physiology in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta)

We investigated intra- and interspecific variation in swimming performance and related physiological parameters in two members of the salmonid family. For our comparisons, we sourced juvenile brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) from one hatchery and a second strain of rainbow trout from another. The hatcheries maintain genetic stocks obtained several decades ago from very different environments. We tested competing hypotheses: that there would be greater interspecific (across species) variation or that there would be greater intraspecific (within species) variation, owing to regional adaptations. To test these hypotheses, individual and small schools of five fish were taken to fatigue using the critical swimming speed test (U(crit)), and three post-exercise physiological metrics, packed red cell volume (hematocrit), citrate synthase and lactate dehydrogenase activity, were assessed. The majority of the results in swimming performance and hematocrit support that intraspecific variation was greater than interspecific variation, i.e. the location had a stronger effect than did genus. Variation in lactate dehydrogenase activity supported neither intra- nor interspecific variation as determining factors. In sum, our findings suggest that the performance of different species of salmonids from the same locale can be more similar than those of the same species from different areas.     

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias)

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.