Effect of hypotonic shock on cultured pavement cells from freshwater or seawater rainbow trout gills

The effect of hypotonic shock on cultured pavement gill cells from freshwater (FW)- and seawater (SW)-adapted trout was investigated. Exposure to 2/3rd strength Ringer solution produced an increase in cell volume followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also induced a biphasic increase in cytosolic Ca(2+) with an initial peak followed by a sustained plateau. Absence of external Ca(2+) did not modify cell volume under isotonic conditions, but inhibited RVD after hypotonic shock. [Ca(2+)](i) response to hypotonicity was also partially inhibited in Ca-free bathing solutions. Similar results were obtained whether using cultured gill cells prepared from FW or SW fishes. When comparing freshly isolated cells with cultured gill cells, a similar Ca(2+) signalling response to hypotonic shock was observed regardless of the presence or absence of Ca(2+) in the solution. In conclusion, gill pavement cells in primary culture are able to regulate cell volume after a cell swelling and express a RVD response associated with an intracellular calcium increase. A similar response to a hypotonic shock was recorded for cultured gill cells collected from FW and SW trout. Finally, we showed that calcium responses were physiologically relevant as comparable results were observed with freshly isolated cells exposed to hypoosmotic shock.     

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

Biochemical responses in gills of rainbow trout exposed to propiconazole

The aim of this study is to investigate the toxic effect of PCZ, a triazole fungicide commonly present in surface and ground water, on the ROS defense system and Na₊-K₊-ATPase in gills of rainbow trout exposed to sublethal concentrations (0.2, 50 and 500 μg L⁻¹) for 7, 20 and 30 days. After prolonged exposure of PCZ at higher test concentrations (50 and 500 μg L⁻¹), oxidative stress was apparent as reflected by the significant higher ROS levels in fish gill, as well as the significant inhibition of SOD and CAT activities. In addition, Na₊-K₊-ATPase activities were significantly lower than those of the control with increasing PCZ concentration and prolonged exposure period. The results of this study indicate that chronic exposure to PCZ has altered multiple physiological indices in fish gill; however, before these parameters are used as unique biomarkers for monitoring residual pharmaceuticals in aquatic environments, more detailed laboratory experiments need to be performed.     

Effects of iron on rainbow trout gill cells in primary culture

This study investigated the effects of iron in the form of iron sulphate (FeSO₄·7H₂O), over the range 0.01–1 mM on rainbow trout primary gill cells cultured on semi-permeable membranes. The endpoints measured were cell proliferation, mucous cell numbers, area of mucus in mucous cells, ultrastructural analysis and transepithelial resistance. Regardless of the concentration, FeSO₄ did not modify the apical surface of pavement cells (microridge) and mucous cells. However, at 1 mM, this metal reduced cell numbers, by inhibiting cell proliferation and causing cell death, and induced a decrease in transepithelial resistance. It is interesting to note that cell numbers were also reduced in the presence of 0.5 mM iron salt, although this reduction did not modify transepithelial resistance. FeSO₄ reduced mucous cell number but did not change mucus area in mucous cells suggesting that this metal could induce a discharge of mucous cells, but mucus secretion would be total and not partial. In conclusion, our in vitro model has allowed to study some toxic effect but also resistance of gill epithelium in presence of iron.     

Evidence for a non-respiratory intralamellar shunt in perfused rainbow trout gills

• 1.1. Rainbow trout (Salmo gairdneri Rich.) heads were perfused with phosphate buffered saline containing 10 μM adrenaline. The gills were ventilated with aerated water (PI_O2 = 20.7–20.9 kPa). Pv_O2 and Pa_O2 were measured and PI_O2 controlled.
• 2.2. An equilibrium between PI_O2 and Pa_O2 when Pv_O2 = 0 kPa, was never observed. The reasons for this difference in O₂ tensions were evaluated.
• 3.3. Perfusion with nitrogen equilibrated saline (Pv_O2 = 0 kPa) resulted in Pa_O2 of 16.4 ± 0.4 kPa. After a change to aerated saline (Pv_O2 20.7–20.9 kPa) Pa_O2 increased to 17.6 ± 0.5 kPa. In this situation when Pv_O2 = PI_O2 the PI_O2 − Pao, difference showed that gill tissue consumed O₂ from the saline.
• 4.4. As expected, an inhibition of this O₂ consumption with cyanide in the ventilation water increased Pa_O2 to 20.1 ± 0.3 kPa. However a return to the N₂-equilibrated saline (Pv_O2 = 0 kPa), still in presence of cyanide, decreased Pa_O2 to 17.2 ± 0.5 kPa. This indicates that O₂ consumption of gill tissue is not the only factor limiting O₂- transfer.
• 5.5. An increase of perfusion flow (Q) within a physiological flow rate from 0.5 to 3.0 ml min⁻¹ (100 g fish)⁻¹ did not significantly affect Pa_O2 Thus, the O₂ transfer in the perfused gills appear to be rather limited by perfusion than by diffusion.
• 6.6. Ventilation at the rate used (800–1000 ml min⁻¹ (100 g fish)⁻¹)did not limit O₂-transfer. The PI_O2–Pa_O2 difference was not caused by a “water shunt”.
• 7.7. We suggest therefore, that the PI_O2-Pa_O2 difference in saline perfused gills is a result of a shunt, i.e. perfusate flow by-passing the gas exchange area in the gill circulation. The shunt was calculated to 11–22 of the total perfusion flow (Q).

     

Insulin affects ionic composition of rainbow trout erythrocytes

As is the case with the anucleate mammalian erythrocyte, ionic composition in anucleate red cells of rainbow trout, Salmo gairdneri, is insulin-sensitive. By comparison with erythrocytes cultured in insulin-free medium, those exposed to insulin concentrations of 20, 200 and 2000 microU/ml for 2 and 6 h exhibited dose-dependent increases in potassium and water content coupled with reductions in the levels of sodium, magnesium and chloride. These observations suggest that the membrane of this type of erythrocyte possesses insulin receptors.     

Endothelin redistributes blood flow through the lamellae of rainbow trout gills

The lamellae of the fish gill are the primary sites for oxygen uptake from the water. Here, only two very thin layers of cells separate the blood from the water. Therefore, energetically costly ion-fluxes will also occur between blood and water, and it has been hypothesised that the blood flow within the lamellae can be regulated through vasoconstriction, but evidence for this has been lacking. Through direct observations of the lamellae of rainbow trout (Oncorhynchus mykiss) in vivo, using epi-illumination microscopy, we show here that an endothelium-derived vasoactive peptide, endothelin-1 (ET-1, 0.2 μg kg⁻¹ or 1.0 μg kg⁻¹), is able to completely constrict the vascular sheet in the lamellae, probably by inducing contraction of pillar cells. This coincided with a dose-dependent increase in ventral aortic blood pressure (rising from 6.6 kPa to 12.0 kPa in response to the high ET-1 dose). However, blood continued to flow through the marginal channel that circumvents each lamella. Thus, ET-1 caused an intralamellar blood shift from the lamellar sheet towards the marginal channels. Vasoconstriction in the lamellae is likely to provide the fish with a mechanism for matching its respiratory surface area with its respiratory needs, thereby minimising ion-fluxes. Accepted: 8 September 1998     

Domestication shapes morphology in rainbow trout Oncorhynchus mykiss

In this study, clonal lines from North American resident and migratory populations of rainbow trout Oncorhynchus mykiss adapted to different geographical conditions and with different domestication histories were characterized morphologically. Lines reared in a common‐garden experiment were characterized for external shape and meristic values, searching for a general pattern of morphological variation due to exposure to captive conditions. A sharp distinction was identified between wild and captive lines. The body profile was deeper in captive lines, with longer dorsal and anal fins and shorter and deeper caudal peduncles. Highly significant differences were also identified in meristic values among the lines but no consistent relation between meristic values and domestication status was detected. This morphological characterization will facilitate the selection of lines with divergent phenotypes for subsequent quantitative trait loci analysis, aimed at identifying genome regions linked with morphological adaptive response to captive conditions.     

Salinity acclimation affects the somatotropic axis in rainbow trout

In this study, we set out to examine the role of the somatotropic axis in the ion-regulation process in rainbow trout. Specifically, our objective was to examine whether plasma insulin-like growth factor-binding proteins (IGFBPs) are modulated by gradual salinity exposure. To this end, freshwater (FW)-adapted rainbow trout were subjected to gradual salinity increases, up to 66% seawater, over a period of 5 days. During this acclimation process, minimal elevations in plasma Ca2+ and Cl- were seen in the salinity-acclimated groups compared with FW controls. There were no changes in plasma Na+ levels, and only a minor transient change in plasma cortisol levels was seen with salinity exposure. The salinity challenged animals responded with elevations in plasma growth hormone (GH) and IGF-I levels and gill Na+-K+-ATPase activity. We identified IGFBPs of 21, 32, 42, and 50 kDa in size in the plasma of these animals, and they were consistently higher with salinity. Despite the overall increase in IGFBPs with salinity, transient changes in individual BPs over the 5-day period were noted in the FW and salinity-exposed fish. Specifically, the transient changes in plasma levels of the 21-, 42-, and 50-kDa IGFBPs were different between the FW and salinity groups, while the 32-kDa IGFBP showed a similar trend (increases with sampling time) in both groups. Considered together, the elevated plasma IGFBPs suggest a key role for these binding proteins in the regulation of IGF-I during salinity acclimation in salmonids.     

Stress responsiveness affects dominant-subordinate relationships in rainbow trout.

The magnitude by which plasma cortisol levels increase following exposure to a stressor is a heritable trait in rainbow trout. The relative growth in coculture of F1 lines selected for high responsiveness (HR) and low responsiveness (LR) to a confinement stressor suggested that behavioral characteristics related to food acquisition, aggression, or competitive ability might differ between the two lines. This hypothesis was tested using the F2 generation of the selected lines. The F2 lines clearly exhibited the characteristics of the F1 parents, displaying significantly divergent plasma cortisol responses to a 1-h confinement stressor and a high heritability for the trait. Behavioral differences between the lines were assessed by observing the outcome of staged fights for dominance in size-matched pairs of HR and LR fish. The identification of dominant and subordinate fish within each pair on the basis of their behavior was supported by the levels of blood cortisol in the fish attributed to each group (dominant < subordinate). Fish from the LR line were identified as dominant in significantly more trials than were HR individuals. The results suggest that behavioral attributes that affect the outcome of rank-order fights are closely linked to the magnitude of the plasma cortisol response to stress in rainbow trout. Whether the link is causal or circumstantial is not yet evident.     

Creatine supplementation affects sprint endurance in juvenile rainbow trout

Fingerling rainbow trout were supplemented with equal amounts of creatine (Cr) by two routes: dietary (12.5 mg Cr per g food); or intraperitoneal injection (0.5 mg Cr per g fish). Endurance in a fixed velocity sprint test (at a speed of 7 BL s(-1)), and resting levels of white muscle metabolites (total creatine [a measure of free creatine plus phosphocreatine (PCr), ATP, lactate and glycogen] were assessed following 7 days of supplementation and compared to controls. None of the treatments had a significant effect on growth, muscle total creatine, percent phosphorylation of creatine, ATP or lactate. However, resting muscle glycogen was elevated in creatine-supplemented fish. Higher muscle glycogen corresponded to significantly greater endurance in creatine-supplemented fish. Although fish do not actively transport additional creatine into the muscle, a mechanism whereby circulating creatine acts to enhance muscle glycogen is present. These results suggest that the improved endurance may be due to an insulin-dependent mechanism (similar to that elucidated in mammalian studies) that allows fish to supercompensate muscle glycogen stores, thus extending endurance through enhanced glycolytic flux.     

Ventilation in rainbow trout (Salmo gairdneri, Richardson) with damaged gills

An account is given of damage to the gills of trout which involved reduction in total filament length and consequently reduced respiratory surface area. It was noted that fish with the greatest gill damage tended to ventilate the gills by active swimming (ram ventilation) to a greater extent than those with more normal gills.     

Effects of hypoxia on isolated vessels and perfused gills of rainbow trout.

Local hypoxia dilates systemic and constricts pulmonary blood vessels in mammals without neural or humoral involvement. The direct effects of hypoxia on isolated vessels from bony fish have not been examined. In the present study, isolated vessels (efferent branchial artery, EBA; coeliacomesenteric artery, CMA; ventral aorta, VA; and anterior cardinal vein, ACV) from rainbow or steelhead trout (Oncorhynchus mykiss) were subjected to either passive load (resting tension) or contracted with a ligand or 50 mM KCl and then subjected to 60 min of hypoxia by N(2) administration and an additional 30 min of normoxia. All vessels were usually refractory to hypoxia under conditions of resting tension. EBAs, CMAs and VAs pre-contracted with a receptor-mediated ligand were all significantly relaxed by hypoxia and only VAs recovered significantly upon subsequent restoration of normoxia. In contrast, tension in all arteries pre-contracted with 50 mM KCl was elevated further in response to hypoxia. Conversely, ligand-contracted ACVs responded to hypoxia with a further increase in tension, whereas KCl-contracted ACVs relaxed. During apparently random 2-3-week periods EBA and CMA from steelhead and EBA from rainbow trout were hyper-reactive to hypoxia. Steelhead vessels responded to hypoxia with a rapid contraction that increased in magnitude over 3 days. These contractions were independent of pre-stimulation and they were dose-dependent upon PO(2). In isolated gills, hypoxic perfusate produced an immediate but transient elevation of resistance (R(GILL)) in all four gill arches. R(GILL) increased by as much as 30% of initial values and this response was unaltered upon a second hypoxic exposure. These studies demonstrate that isolated vascular segments of rainbow trout are indeed responsive to hypoxia and that these differential responses are vessel and tone dependent and the overall response may be altered by as yet unknown seasonal or environmental factors. Hypoxia-induced arterial relaxation is blocked by elevated external [K(+)], implicating alteration of transmembrane K(+) conductance and/or membrane potential in this depressor response. K(+)-channel closure or voltage-gated Ca(2+) influx cannot account for arterial vasoconstriction due to hypoxia during KCl contractions. Vascular responses to hypoxia could have a profound impact on local flow in vivo and could mediate ventilation-perfusion matching in the branchial circulation of fish.