Comparison of angiotensin converting enzyme-like activity in the Antarctic teleosts <Emphasis Type="Italic">Trematomus bernacchii</Emphasis> and <Emphasis Type="Italic">Chionodraco hamatus</Emphasis>

Biochemical parameters of the angiotensin converting enzyme-like activity (ACELA) in the gills of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii were characterized. Enzymatic activity was revealed following hydrolysis of a specific substrate of angiotensin-converting enzyme N-[3-(2-furyl)acryloyl]l-phenylalanyl-glycyl-glycine (FAPGG) and metabolites were separated by reverse phase HPLC analysis. The results showed similar Km values for the substrate FAPGG at 5°C for the two species with an increase of Km value for T. bernacchii at 25°C. The optimum pH value was 8.5 at 25°C and optimum chloride concentrations were about 300 mM. In T. bernacchii the optimum temperature for maximum enzyme activity was 50°C, while maximum activity in C. hamatus occurred at 35°C. Lisinopril was more efficient in inhibiting ACELA in C. hamatus with an I ₅₀ value of 16.83 ± 5.11 nM, compared to an I ₅₀ value of 30.66 ± 5.19 nM in T. bernacchii. In conclusion, it appears that some biochemical parameters of ACELA in C. hamatus differ from those in T. bernacchii, probably due to different ways that the enzyme adapts to the constantly cold temperatures of the animal’s environment.     

Relationship between metallothionein and metal contents in red-blooded and white-blooded Antarctic teleosts

Metal bioaccumulation and metallothionein were investigated in different organs of the red-blooded teleost, Trematomus bernacchii and the haemoglobinless Chionodraco hamatus. Specimens of the two Antarctic fish were sampled from Terra Nova Bay (Ross Sea), and their levels of Cd, Cu and Zn in homogenates and in soluble fractions of liver, muscle, gills, heart and plasma were determined. Dosages of metallothioneins (MTs) or MT-like proteins were assayed by the silver saturation method in soluble fractions of the same organs. In both T. bernacchii and C. hamatus the highest MT contents were found in liver. Hepatic MT and Cd, Cu and Zn concentrations correlated positively in T. bernacchii, whereas in C. hamatus hepatic MT showed a positive correlation only with Cd. Positive correlations were also found between Cd and MT in gills of the two species. A metal-binding protein containing a high percentage of cysteine from C. hamatus was purified and compared with the MT from T. bernacchii and mammals. Accepted: 27 November 1999     

Mitochondria-rich cells in Antarctic fish gills

We used transmission and scanning electron microscopy and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills of two Antarctic fishes, Chionodraco hamatus (Channichthyidae) and Trematomus bernacchii (Nototheniidae). The general morphology of the gills in the two species was slightly different: in T. bernacchii the filament and secondary lamellae were short and thicker, and mitochondria-rich cells were less numerous than in C. hamatus. In the two species the mitochondria-rich cells, distributed in the secondary and primary epithelium, were of the same morphological type, similar to the α-chloride cells of temperate seawater teleosts. The study was carried out to compare the mitochondria-rich cells of Antarctic fishes to the chloride cells of temperate marine teleosts. Immunolocalisation, using a specific antibody to the α-subunit of Na⁺/K⁺-ATPase, was observed in numerous epithelial cells in the interlamellar epithelium and on the secondary lamellae, suggesting an active sodium chloride secretion. Accepted: 16 October 1999     

Induction of mixed function oxidase (MFO) system in two species of antarctic fish from Terra Nova Bay (Ross Sea)

Summary The aim of this study was to investigate the detoxicant Mixed Function Oxidase system in two species of Antarctic fish, C. hamatus and P. bernacchii, collected during the Antarctic summer of 1989–1990 at the Italian Scientific Station at Terra Nova Bay (Ross Sea). Several specimens were induced by injection of phenobarbital (PB), 3-methylcholanthrene (3-MC) and PCBs in the caudal vein. The results show significant differences between the two species. In C. hamatus, basal activity of benzo(a)pyrene monooxygenase (BPMO) was among the lowest measured even in fish of temperate seas, and in P. bernacchii it was 20 times lower. The values of regenerating activities (NADPH-cytochrome c reductase (NADPH-CYTCRED), NADH-cytochrome c reductase (NADH-CYTCRED) and NADH-ferricyanide reductase (NADH-FERRIRED) suggest that the two species use different electron donor molecules. Injection of chemicals in the caudal vein did not provoke induction of MFO activity in P. bernacchii. In C. hamatus, phenobarbital and PB-type inducers did not cause induction but there was a statistically significant response to 3-MC.     

Spermatogenesis in some Antarctic teleosts from the Ross Sea: histological organisation of the testis and localisation of bFGF

Testis structure and spermatogenetic activity were studied in two Antarctic teleostean species, Chionodraco hamatus and Trematomus bernacchii, captured during the austral summer in the Ross Sea. The specimens of C. hamatus showed full reproductive activity but, the spermatogenetic cycle being over, only spermatogonia and Sertoli cells were present in the seminiferous tubules whereas the lumina were full of sperm. By contrast, the specimens of T. bernacchii were in the stage of spermatogenetical recrudescence, having not yet entered the reproductive period. In this species, the seminiferous tubules were devoid of lumen and full of spermatogonial cysts, showing some mitoses. Many tubules contained cysts of meiotic spermatocytes I and, in one case only, small cysts of spermatocytes II. The final stages of spermatogenesis were lacking, presumably occurring later, in autumn/winter. The immunocytochemical tests aimed at identifying bFGF and FGFR1 revealed a positive reaction both in Sertoli cells and spermatogonia in the C. hamatus specimens, indicating that this species was ready to start a new spermatogenetic cycle. The weak reaction in the specimens of T. bernacchii suggests that, in this species, the stage of cell division was over and that of meiosis and differentiation was starting. These data indicate that Antarctic fish have an opportunistic spermatogenetic cycle. Accepted: 24 October 1999     

Renin-angiotensin system in antarctic fishes

• 1.1. The presence of a renin-angiotensin-like system has been investigated in the Antarctic fishes Chionodraco hamatus (Fam. Channichthydae) and Pagothenia (Trematomus) bernacchii (Fam. Notothenidae).
• 2.2. A renin-like activity is present in plasma and kidney of both the white blooded (Chionodraco) and the red blooded (Pagothenia) species.
• 3.3. An angiotensin converting enzyme-like activity has been demonstrated in plasma, gills and kidneys of both species. The activity is inhibited by high temperature.
• 4.4. From our data a renin-angiotensin-like system is present in the Antarctic fishes studied but the cascade of enzymes is active only at low temperatures.

     

Effects of warm acclimation on serum osmolality,cortisol and hematocrit levels in the Antarctic fish, <Emphasis Type="Italic">Trematomus bernacchii</Emphasis>

Antarctic fish, such as the Trematomus bernacchii, living at −1.9°C maintain a serum osmolality of around 600 mOsm kg⁻¹, nearly twice that of temperate fish. Upon warm acclimation, Antarctic fish significantly lower their serum osmolality. It has been suggested that this response to warm acclimation is due to stress. The purpose of this study was to determine, whether upon warm acclimation there was a change in the levels of the stress hormone cortisol and hematocrit associated with the decrease in serum osmolality. T. bernacchii were warm acclimated up to 4 weeks and serum osmolality, cortisol and hematocrit were measured. Upon warm acclimation to +1.6 and +3.8°C over the course of 4 weeks, T. bernacchii significantly lowered their serum osmolality (from 547 ± 4 mOsm kg⁻¹ to 494 ± 6 and 489 ± 4 mOsm kg⁻¹, respectively), yet did not alter their serum cortisol (29 ± 6 nl ml⁻¹) or hematocrit (22 ± 1%) levels. These results suggest that warm acclimation does not induce a stress response in T. bernacchii.     

Blood Lipids in Antarctic and in Temperate-Water Fish Species

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 ω-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, ω-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.     

Regulatory peptides in the urinary bladder of two genera of Antarctic teleosts

Somatostatin 14, prolactin, atrial natriuretic peptide, galanin and urotensin II were found using immunohistochemistry in the urinary bladders of the Antarctic fishes Trematomus bernacchii (Nototheniidae) and Chionodraco hamatus (Channichthyidae) caught in the Ross Sea. The urinary bladders of the two species showed a different histology in the epithelial layer. In T. bernacchii the epithelium comprises a single type of columnar cells, while in C. hamatus the columnar cells are restricted to the ventral portion of the bladder, and the dorso-lateral region is lined by cuboidal cells. No difference in the intensity of the immunostaining was observed in the two cell types; the only variation was a different distribution of the immunoreactions, which were present in the whole cytoplasm in the cuboidal cells and restricted to the apical and/or basal portion of the columnar cells. Accepted: 1 November 1998     

Ion transport systems in the kidney and urinary bladder of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii

Na⁺-K⁺-Cl^- cotransport and Na⁺/K⁺ATPase were studied by immunohistochemistry in the kidney and urinary bladder of Trematomus bernacchii and Chionodraco hamatus. The activity was correlated to the density of mitochondria. The first segment of the renal proximal tubule was more active than the second one. In T. bernacchii and the temperate marine teleost Pagellus bogaraveo, the immunoreactivity for the antibody to cotransporters and to the !-subunit of the sodium pump was stronger than in the icefish. This difference indicates in the kidney of the icefish, a weaker secretory activity, a consequent lower osmolarity in the lumen and lower water loss, which correlates well with the need for a greater blood volume in the icefish. The epithelium of the urinary bladder in T. bernacchii, where intense immunostaining was observed, was composed of columnar cells. In C. hamatus the columnar cells, where the immunostaining was weaker, lined only a portion of the urinary bladder, the other region being composed of cuboidal cells.     

Qualitative and quantitative analysis of serum immunoglobulins of four Antarctic fish species

Immunoglobulins from the Antarctic fish species Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi, and Chionodraco hamatus were analysed in whole serum and after purification by affinity chromatography on protein A-sepharose. Using SDS-PAGE, the apparent masses of the heavy and light chains were, respectively, 83.5 kDa and 27.5 kDa for T. bernacchii, 83.5 kDa and 27 kDa for T. hansoni, 81 kDa and 27.5 kDa for T. newnesi, and 74.5 kDa and 30 kDa for C. hamatus. It was not possible to purify immunoglobulins from T. newnesi due to their low concentration in serum. Heterogeneity in mass of both heavy and light chains was observed in all species. By using a polyclonal antibody raised against sea bass immunoglobulins, cross-reactivity was observed with heavy and light chains of all species. With this antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and results showed the relative immunoglobulin concentration in sera of the Antarctic fish species considered, using as standard sea bass immunoglobulins. Received: 25 November 1996 / Accepted: 3 February 1997     

Effects of temperature on mitochondrial function in the Antarctic fish Trematomus bernacchii

Effects of temperature on O₂ consumption by mitochondria of the Antarctic fish Trematomus bernacchii were compared with effects obtained with mitochondria from tropical (Sarotheridon mossambica) and temperate zone fishes (Sebastes carnatus and Sebastes mystinus). Arrhenius plots of O₂ consumption versus temperature exhibited slope discontinuities (“breaks”) at temperatures (Arrhenius break temperatures: ABTs) reflective of the species' adaptation temperatures. The ABT for mitochondria of T. bernacchii is the lowest reported for any animal and is ∼12 °C below the value predicted by a regression equation based on ABT data for several invertebrates and fishes. The temperature at which the acceptor control ratio (ACR), an index of efficiency of coupling of electron transport to synthesis of ATP, began to decrease with rising temperature also reflected adaptation temperature. The decrease in ACR with rising temperature began at ∼18 °C for mitochondria of T. bernacchii, in contrast to ∼35 °C for mitochondria of Sarotheridon mossambica. Maintaining T. bernacchii at 4 °C for 2 weeks led to no changes in ABT or in the response of ACR to temperature. The thermal sensitivities of mitochondria of T. bernacchii reflect the high level of cold adaptation and stenothermy that is characteristic of Antarctic Notothenioid fishes. Accepted: 5 January 1998     

Antarctic teleost immunoglobulins: more extreme, more interesting

We have investigated the immunoglobulin molecule and the genes encoding it in teleosts living in the Antarctic seas at the constant temperature of −1.86 °C. The majority of Antarctic teleosts belong to the suborder Notothenioidei (Perciformes), which includes only a few non-Antarctic species. Twenty-one Antarctic and two non-Antarctic Notothenioid species were included in our studies. We sequenced immunoglobulin light chains in two species and μ heavy chains, partially or totally, in twenty species. In the case of heavy chain, genomic DNA and the cDNA encoding the secreted and the membrane form were analyzed. From one species, Trematomus bernacchii, a spleen cDNA library was constructed to evaluate the diversity of VH gene segments. T. bernacchii IgM, purified from the serum and bile, was characterized. Homology Modelling and Molecular Dynamics were used to determine the molecular structure of T. bernacchii and Chionodraco hamatus immunoglobulin domains. This paper sums up the previous results and broadens them with the addition of unpublished data.     

Drinking in Antarctic fishes

Drinking rates have never been measured in Antarctic fish. Drinking rates were measured in four species of notothenioid fish, including a hemoglobinless icefish, found in the near-freezing waters of the Ross Sea of the Southern Ocean. All of the fish, with the exception of the icefish, had low drinking rates and high serum osmolalities relative to temperate seawater fish. The icefish had significantly higher drinking rates and serum osmolalities relative to the Antarctic fishes containing hemoglobin, including Trematomus bernacchii. Warm acclimation of T. bernacchii, from −1.5°C to +4°C for 4 weeks, significantly increased their drinking rates 4.6-fold, significantly decreased their serum and intestinal osmolality by 11% and 12%, respectively, relative to cold-acclimated fish. These results indicate that increased drinking rates in Antarctic fish at elevated temperatures are involved in maintaining a lower serum osmolality.     

Characterization of the kallikrein-kinin system during the bovine ovulation process

The kallikrein–kinin system (KKS) has been described as an important mediator of physiologic processes. Kallikreins use kininogen (KNG) as substrate to generate bradykinin, the main active peptide of the KKS that acts through two types of receptors, the B₁R and the B₂R. The goal of this study was to characterize some components of the KKS in different compartments of the ovary during the bovine ovulation process. The KNG, B₁R and B₂R mRNA expression patterns were assessed in theca and granulosa cells, as well as the bradykinin concentration and kallikrein-like activity in follicular fluid of bovine periovulatory follicles. To obtain a periovulatory follicle (≥12 mm), twenty-seven cows were submitted to estrus synchronization protocol and ovariectomized by colpotomy at 0, 3, 6, 12 or 24 h after a GnRH-analog injection (gonadorelin; 100 μg, IM). Follicular fluid was aspirated for enzymatic assays while granulosa and theca cells were harvested for mRNA analysis. The mRNA expressions in follicular cells were evaluated by real-time RT-PCR and data representation related to the cyclophilin housekeeping gene. The bradykinin concentration and kallikrein-like activity were measured in follicular fluid by enzymatic immunoassay and selective substrate cleavage, respectively. The B₂R expression in theca cells and B₁R expression in theca and granulosa cells showed different profiles during the periovulatory period (P < 0.05). The bradykinin concentration and kallikrein-like activity in the follicular fluid were different (P < 0.05) due to the time during the ovulation process. KNG mRNA expression was similar for both follicular cell types (P > 0.05). Taken together, these results provide an important characterization of the presence and possible KKS regulation during the bovine ovulation.     

Thin-blooded Antarctic fishes: a rheological comparison of the haemoglobin-free icefishes Chionodraco kathleenae and Cryodraco antarcticus with a red-blooded nototheniid, Pagothenia bernacchii

The icefishes (family Channichthyidae) comprise a unique group of teleost fishes endemic to Antarctic and sub-antarctic seas. All members of the family totally lack haemoglobin. Haematological parameters and viscosity were determined for blood from 11 specimens of two channichthyid species (Chionodraco kathleenae Regan, 1914; Cryodraco antarcticus Dollo, 1900), and 14 specimens of a red-blood Antarctic nototheniid species (Pagothenia bernacchii (Boulenger, 1902)), captured near the Italian research station at Terra Nova Bay, Ross Sea, Antarctica. Channichthyid blood contained only a small number of non-pigmented cells (10 000-40 000 cells μI^?1, depending on species) in contrast to nototheniid blood (360 000-450 000 cells μI^?1 in unstressed specimens). Blood viscosity was measured by cone plate viscometry over a range of shear rates (11.3-450s ^?1), at six temperatures between – 1.8°C and + 15°C. At the ambient Antarctic seawater temperature of – 1.8° C, and at low shear rate (22.5 s^?1), the viscosity of channichthyid blood was relatively low (3.99 ± 0.40 cP) compared with blood taken from unstressed P. bernacchii, which was about 25% more viscous (4.91 ± 0.59 cP). The viscosity of channichthyid blood was almost independent of shear rate, approximating an ideal Newtonian fluid, while the viscosity of nototheniid blood was much more dependent upon both shear rate and temperature, increasing sharply at low shear rates and low temperatures. Viscosity of nototheniid blood varied with haematocrit, which was in turn strongly influenced by stress. Blood samples taken from P. bernacchii under moderate stress induced by handling during acute caudal venepuncture had haematocrit values in the range 15–20% and viscosities of 8-l0cP, while undisturbed specimens sampled through a venous cannula yielded haematocrits of 8–10%. The viscosity of nototheniid plasma did not differ significantly from that of channichthyid whole blood or channichthyid plasma. The higher viscosity of nototheniid blood is attributable to cell content, and in stressed specimens possibly also to adrenergic swelling of erythrocytes. The absence of erythrocytes in channichthyid blood avoids the great increases in viscosity which are induced in corpusculate blood by sub-zero seawater temperatures.     

Catecholamine release in heat-stressed Antarctic fish causes proton extrusion by the red cells

Two species of Antarctic fish were stressed by moving them from seawater at −1 °C to seawater at 10 °C and holding them for a period of 10 min. The active cryopelagic species Pagothenia borchgrevinki maintained heart rate while in the benthic species Trematomus bernacchii there was an increase in heart rate. Blood pressure did not change in either species. Both species released catecholamines into the circulation as a consequence of the stress. P. borchgrevinki released the greater amounts, having mean plasma concentrations of 177 ± 54 nmol · l⁻¹ noradrenaline and 263 ± 131 nmol · l⁻¹ adrenaline at 10 min. Plasma noradrenaline concentrations rose to 47 ± 14 nmol · l⁻¹ and adrenaline to 73 ± 28 nmol · l⁻¹ in T. bernacchii. Blood from P. borchgrevinki was tonometered in the presence of isoprenaline. A fall in extracellular pH suggests the presence of a Na⁺/H⁺ antiporter on the red cell membrane, the first demonstration of this in an Antarctic fish. Treatment with the β-adrenergic antagonist drug sotalol inhibited swelling of red blood cells taken from temperature-stressed P. borchgrevinki, suggesting that the antiporter responds to endogenous catecholamines. Accepted: 22 January 1998     

Effects of temperature acclimation on cardiorespiratory performance of the Antarctic notothenioid Trematomus bernacchii

Notothenioid fishes of the Southern Ocean have evolved under cold and stable temperatures for millions of years. Due to rising temperatures in the Southern Ocean, investigating thermal limits and the capacities for inducing a temperature acclimation response in notothenioids has become of increasing interest. Here, we investigated effects of temperature acclimation on cardiorespiratory responses and cardiac and skeletal muscle energy metabolism in a benthic Antarctic notothenioid, Trematomus bernacchii. We acclimated specimens to ?1, 2 and 4.5 °C for 14 days and quantified heart rates and ventilation rates during an acute increase in temperature. Ventilation rates showed an effect of acclimation both at initial steady-state acclimation conditions and during an acute temperature increase, suggesting a partial thermal compensatory response. However, acclimation did not affect heart rates at steady-state acclimation conditions and the temperatures at which onset of cardiac arrhythmia occurred, suggesting lack of inducible thermal tolerance in cardiac performance. Citrate synthase (CS), lactate dehydrogenase (LDH) and 3-hydroxyacyl dehydrogenase activities in skeletal muscle tissues suggested acclimation-induced shifts in metabolic fuel preferences, and a marked increase in LDH activity with acclimation to 4.5 °C showed an increase in anaerobic metabolism. In heart tissue, CS and LDH activities decreased with acclimation to 4.5 °C, suggesting reduced cardiac ATP production. Overall, the data suggest a partial acclimatory response to temperature by T. bernacchii and support the hypothesis that reduced cardiac acclimatory capacity may play a role in limiting the thermal plasticity of T. bernacchii.