Voltage-dependent calcium channels in ventricular cells of rainbow trout: effect of temperature changes in vitro

Voltage-dependent calcium channels (VDCC) in ventricular myocytes from rainbow trout (Oncorhynchus mykiss) were investigated in vitro using the perforated patch-clamp technique, which maintains the integrity of the intracellular milieu. First, we characterized the current using barium as the charge carrier and established the doses of various pharmacological agents to use these agents in additional studies. Second, we examined the current at several physiological temperatures to determine temperature dependency. The calcium currents at 10 degrees C (acclimation temperature) were identified as L-type calcium currents based on their kinetic behavior and response to various calcium channel agonists and antagonists. Myocytes were chilled (4 degrees C) and warmed (18 and 22 degrees C), and the response of VDCC to varying temperatures was observed. There was no significant dependency of the current amplitude and kinetics on temperature. Amplitude decreased 25-36% at 4 degrees C (Q(10) approximately 1.89) and increased 18% at 18 degrees C (Q(10) approximately 1.23) in control, Bay K8644 (Bay K)-, and forskolin-enhanced currents. The inactivation rates (tau(i)) did not demonstrate a temperature sensitivity for the VDCC (Q(10) 1.23-1. 92); Bay K treatment, however, increased temperature sensitivity of tau(i) between 10 and 18 degrees C (Q(10) 3.98). The low Q(10) values for VDCC are consistent with a minimal temperature sensitivity of trout myocytes between 4 and 22 degrees C. This low-temperature dependency may provide an important role for sarcolemmal calcium channels in adaptation to varying environmental temperatures in trout.     

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.     

Force frequency relation in the myocardium of rainbow trout

Summary Isolated heart ventricular preparations from rainbow trout were electrically stimulated to contraction. Following a temporary change in stimulation rate from 0.2 Hz to a higher value, the force fell to a minimum after which it increased and levelled off. Upon the return to 0.2 Hz a further transient increase in force appeared. The latter two responses were stimulated by an increased extracellular K⁺, which is known to inactivate the Na⁺ channel. The initial negative inotropic effect, in contrast to the two subsequent positive effects, was associated with a parallel decrease in amplitude of the action potential measured in 15 mM K⁺, used as an index of the Ca²⁺ influx. One micromolar (1 M) ryanodine did not affect either the negative or the positive responses due to an increase in stimulation rate, but depressed the force developed after prolonged periods of rest. Ten micromolar (10 M) adrenaline strongly inhibited the positive effects of an elevation of frequency. An elevation of extracellular Na⁺ from 141 to 166 mM had a similar effect. In conclusion, the positive effects occurring in 15 mM K⁺ do not seem to depend on the initial Na⁺ current. They may nevertheless depend on changes of the cellular Na⁺ balance as suggested by the effects of adrenaline, K⁺ and Na⁺. The functional role of the sarcoplasmic reticulum is unclear.     

Effects of aluminium and cortisol on in vitro calcium deposition on otoliths in rainbow trout

Aluminium (1–100 μM) reduced in vitro calcium deposition on otoliths concentration-dependently in rainbow trout, but cortisol (1 and 10 μg ml⁻¹) had no effect.     

Influence of temperature on calcium sensitivity in the ventricular myocardium of the South American lungfish: Effects of extracellular calcium and adrenaline

The mechanical properties of ventricular myocardium of the South American lungfish, Lepidosiren paradoxa, acclimated to 25 °C, were evaluated in vitro at 15, 25 and 35 °C. The inotropism (Fc—% of initial values) of ventricle strips was examined in response to adrenaline (from 10⁻⁸ to 10⁻⁵ M) and extracellular calcium (from 2.5 to 14.5 mM) in all experimental temperatures. At 15 and 25 °C, Fc rose when extracellular Ca²⁺ or adrenaline were increased, while Fc remained unchanged at 35 °C. These results suggest that at lower temperatures Ca²⁺ availability is a limiting step to cardiac performance and can be ameliorated by adrenergic stimulation. In contrast, since inotropic agents failed to increase cardiac inotropism at 35 °C, lungfish myocytes seem to show a high temperature sensitivity, which increases Ca²⁺-buffering capacity and/or Ca²⁺ transportation from and to cytosol as well as myofilaments Ca²⁺ sensitivity.     

Effects of temperature on glucose release and glycogen metabolism in isolated hepatocytes from rainbow trout (Salmo gairdneri)

Endogenous glucose release and glycogen metabolism were investigated in isolated hepatocytes from rainbow trout acclimated to 10 and 20 degrees C. Thermal acclimation did not significantly affect hepatocyte glycogen contents and the rates of glucose release during substrate-free incubations. In both acclimation groups glucose production and glycogen metabolism exhibited clearly different dependencies on assay temperature. It was concluded, that there are different sources of glucose release in the lower and upper temperature range--gluconeogenesis from endogenous precursors at low temperatures and glycogenolysis at high temperatures. This conclusion was supported by experiments with 3-mercaptopicolinic acid, which stimulated glycogen breakdown especially in the low temperature range.     

Influence of temperature on silver accumulation and depuration in rainbow trout

To assess the influence of water temperature on silver uptake, rainbow trout Oncorhynchus mykiss ( c . 50 g; held at 13° C) were exposed to 0·1 μM AgNO₃ in ion‐poor water for 1 week at 4 and 16° C without previous temperature acclimation. To assess the influence of temperature on elimination of previously accumulated Ag, rainbow trout were exposed to 0·1 μM AgNO₃ in ion‐poor water for 1 week at 12° C, then were randomly divided amongst two Ag‐free water containers, differing only in temperature (3 and 16° C), for 2 months. In the uptake study greater accumulation of Ag was seen in the gills, plasma and especially the livers and bile of 'warm' rainbow trout (16° C) compared to 'cold' rainbow trout (4° C), which can be explained by the higher metabolic rates of the warmer fish. In the depuration study there was no net elimination of Ag from the livers and bile but there was biphasic elimination of Ag from the gills and plasma of 'warm' and 'cold' fish, but with few differences between them. This indicated that temperature‐dependent processes were less important in Ag elimination than in Ag uptake. Toxicokinetic modelling of Ag uptake by livers indicated four‐fold greater uptake of Ag by 'warm' rainbow trout compared to 'cold' rainbow trout (one compartment uptake model). Elimination of previously accumulated Ag from the plasma was best fitted by a two compartment rate‐constant based model, with approximately half the plasma Ag load eliminated within 24 h, followed by slower elimination of Ag over 2 months.     

Cortisol stimulates calcium transport across cultured gill epithelia from freshwater rainbow trout

Summary The effect of cortisol on calcium (Ca²⁺) transport across cultured rainbow trout gill epithelia composed of both pavement cells (PVCs) and mitochondria-rich cells (MRCs) was examined. Under symmetrical culture conditions (L15 media apical/L15 media basolateral), cortisol had subtle effects on gill epithelial preparations. Both control and cortisol treated epithelia exhibited Ca²⁺ influx and efflux rates (measured radioisotopically using ⁴⁵Ca) that were approximately balanced, with a slight inwardly directed net Ca²⁺ flux. Ussing flux ratio analysis indicated active Ca²⁺ transport in the inward direction across epithelia bathed symmetrically regardless of hormone treatment. In contrast, under asymmetrical conditions (freshwater apical/L15 media basolateral) control epithelia exhibited active Ca²⁺ transport in the outward direction (basolateral to apical) throughout experiments conducted over a 24-h period, whereas cortisol-treated preparations exhibited active transport in the inward direction (apical to basolateral) during the early stages of an asymmetrical culture period (e.g., T0–6 h) and passive transport during the later stages (e.g., T18–24 h). When soft freshwater (with tenfold lower [Ca²⁺]) was used for asymmetrical culture instead of freshwater, control epithelia developed outwardly directed active Ca²⁺ transport properties, whereas cortisol-treated preparations did not. The results of this study support a hypercalcemic role for cortisol in rainbow trout and demonstrate that treating cultured gill epithelia composed of both PVCs and MRCs with cortisol can stimulate active Ca²⁺ uptake under circumstances that more closely resemble natural conditions for fish gills (i.e., freshwater bathing the apical side of the epithelium).     

A pepsinogen from rainbow trout

1. A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach, has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.     

Effect of temperature and temperature acclimation on the ryanodine sensitivity of the trout myocardium

The influence of acute temperature change and temperature acclimation on the sensitivity of contracture development to ryanodine were examined in the rainbow trout myocardium using two preparations: in vitro isolated ventricular strips and in situ working perfused hearts. Ryanodine effects in vitro were dependent on test temperature (8 and 18 °C), pacing frequency (0.2–1.5 Hz) and acclimation temperature (8 and 18 °C). At a pacing frequency of 0.2 Hz and a test temperature of 18 °C, ryanodine depressed isometric tension development in ventricular strips both from trout acclimated to 8 and 18 °C but the decrease was significantly greater in strips from 8 °C-acclimated trout. No ryanodine effect was observed in either acclimation group at a test temperature of 8°C. The effect of ryanodine in vitro was reduced or lost at pacing frequencies greater than 0.2 Hz and at 0.6 Hz ryanodine depressed tension development at 18 °C only in strips from 8 °C-acclimated trout. Ryanodine did not affect tension development at stimulation rates above 0.6 Hz in any test group. Likewise, ryanodine did not significantly impair cardiac performance of in situ working perfused heart preparations which operated at intrinsic beat frequencies in excess of 0.6 Hz. These results suggest that the sarcoplamic reticulum calcium release channel of the trout myocardium is expressed but is not functionally involved in beat-to-beat regulation of contractility at either (1) low temperature (8 °C), or (2) at routine physiological heart rate (>0.6 Hz). However, under conditions in which involvement of the sarcoplasmic reticulum is observed (18 °C and a heart rate < 0.6 Hz), prior acclimation to low temperature results in either a greater capacity of the sarcoplasmic reticulum to store releasable calcium or an increase in the amount of calcium that is in releasable form.Abbreviations bm body mass - E-C coupling, excitation-contraction coupling - IVS isometric ventricular strip - SR sarcoplasmic reticulum - TES N-tris[hydroxy-methyl]methyl-2-aminoethane sulfonic acid - WPH in situ working perfused heart     

Inotropic effects of adrenaline on the anoxic or hypercapnic myocardium of rainbow trout and eel

Summary The effects of adrenaline on the development of force under anoxia and hypercapnic acidosis (13% CO₂ in 30 mM HCO ₃ ^– ) were examined in isolated, electrically stimulated cardiac ventricle strips of rainbow trout and eel.During anoxia or acidosis applied 15 min in advance, the adrenaline concentration of the bathing solution was increased in 5 steps from 0 to 10^–4 M with 5 min at each step. Before levelling off the contractile tension increased by 145±42% (mean±SE) in the anoxic, 80±14% in the acidotic and 152±41% in the control trout cardiac strips. Except for the acidotic strips the corresponding values tended to be lower for the eel strips being 46±9%, 57±17% and 57±9%, respectively. The inotropic affinity for adrenaline was lower in the trout than in the eel myocardium. For the trout myocardium it remained unchanged, while it decreased somewhat for the eel myocardium under anoxia or acidosis.Adding to the muscle bath 10^–5 M adrenaline resulted in an increase in force development by about 90% for the trout myocardium and 50% for the eel myocardium. 5 min later anoxia or hypercapnic acidosis was applied for 30 min followed by 30 min at control conditions. Relative to the force values recorded just before anoxia or acidosis was applied, the changes in contractile force during these periods were the same with and without adrenaline. Thus adrenaline appears to have a persistent positive inotropic effect in the fish myocardium during and after oxygen lack or acidosis.     

Effects of dilution and temperature of analysis on blood serum values in rainbow trout, Salmo gairdneri

Albumin, cholesterol, glucose, inorganic phosphorus, and total protein analyses were not affected by temperatures between 20 and 38°C nor by dilution with either 0.65% or 0.85% NaCl. Additionally, blood urea nitrogen, calcium and creatinine were unaffected by dilution. Alkaline phosphotase, cholinesterase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, α-hydroxybutyrate dehydrogenase, lactic dehydrogenase, and phosphohexose isomerase were all affected by temperature. Results of dilution tests were generally inconclusive, while results of enzymatic reactions indicate that reaction temperatures must be closely controlled to produce comparable results.     

Effects of triploidy on rainbow trout myogenesis in vitro

To examine the effects of triploidy on rainbow trout myogenesis in vitro , mononuclear cells were liberated enzymatically from the lateralis muscles of diploid and triploid trout. The muscle of diploids yielded 1 × 10⁶± 1 × 10⁵ (± s.e.m. ) mononuclear cells g⁻¹ muscle compared to 0.7 × 10⁶± 8 × 10⁴ cells g⁻¹ from triploids ( P <0.01). The plating efficiencies of diploid and triploid mononuclear cells on Matrigel™ following 18 h of culture in Leibovitz's L-15 + 10% foetal bovine serum were not significantly different, 35.0 ± 3.5% and 33.0 ± 2.9%, respectively. For most time points examined, the proportion of nuclei in multinucleated cells and the proportion of nuclei in myosin positive cells were not significantly different for diploid and triploid trout. Taken together, these data suggest that diploid and triploid myogenic cells will differentiate similarly when compared under identical, in vitro conditions.     

Effects of endothelin-1 and homologous trout endothelin on cardiovascular function in rainbow trout

The cardiovascular effects of endothelin (ET)-1 and the recently sequenced homologous trout ET were examined in unanesthetized trout, and vascular capacitance curves were constructed to evaluate the responsiveness of the venous system to ET-1. A bolus dose of 667 pmol/kg ET-1 doubled ventral aortic pressure; produced a triphasic pressor-depressor-pressor response in dorsal aortic pressure (P(DA)); increased central venous pressure, gill resistance, and systemic resistance; and decreased cardiac output, heart rate, and stroke volume. These responses were dose dependent. Bolus injection of trout ET (333 or 1,000 pmol/kg) produced essentially identical, dose-dependent cardiovascular responses as ET-1. Dorsal aortic infusion of 1 and 3 pmol. kg(-1). min(-1) ET-1 and central venous infusion into the ductus Cuvier of 0.3 and 1 pmol. kg(-1). min(-1) produced similar dose-dependent cardiovascular responses, although the increase in P(DA) became monophasic. The heightened sensitivity to central venous infusion was presumably due to the more immediate exposure of the branchial vasculature to the peptide. Infusion of 1 pmol. kg(-1). min(-1) ET-1 decreased vascular compliance but had no effect on unstressed blood volume. These results show that ETs affect a variety of cardiovascular functions in trout and that branchial vascular resistance and venous compliance are especially sensitive. The multiplicity of effectors stimulated by ET suggests that this peptide was extensively integrated into cardiovascular function early on in vertebrate phylogeny.     

Linking microhabitat availability and local density of rainbow trout in low-gradient Japanese streams

Quantification of reach-based microhabitat availability for rainbow trout (Oncorhynchus mykiss), considering their microhabitat requirements in two low-gradient streams, northern Japan, was attempted to test for habitat space limitation of local trout density. Underwater observations revealed that fish selected microhabitats of moderate current velocity, relatively greater depth and shorter distance to overhead cover in both streams, although habitat features used and available differed slightly between the streams. Habitat space for fish potentially available in the channel environment was evaluated using principal component analysis (PCA) of both available and used microhabitat. A close relationship was evident in both streams between reach-based microhabitat availability and fish density, which was assessed by a three-pass removal method. Direct estimates of fish microhabitat availability using PCA can contribute to accurate predictions of local fish density and provide insight into the mechanisms responsible for fish–habitat relationships in streams.     

Effects of gossypol on rainbow trout Salmo guirdneri Richardson

Rainbow trout were fed gossypol acetate or cottonseed meals containing gossypol for 14 months. Toxic effects were indicated by growth depression, reduced hematocrit, hemo-globin, and total plasma protein. Histological changes were noted, particularly in the liver and kidney.     

Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout

We investigated whether there are compensatory changes in the coronary microvasculature, cardiac lipid metabolism, and myocyte ultrastructure associated with ventricular enlargement in male rainbow trout. Epicardial tissue was sampled at different stages of sexual maturation, and we estimated arterial capillary density, intercapillary diffusion distance, and applied a diffusion model to predict PO₂ at different workloads. We also measured biochemical indices of lipid metabolism and estimated fractional volumes of mitochondria and myofibrils in myocytes. Immature fish with nonenlarged ventricles had the highest capillary length densities (1620±158 mm mm⁻³). Maturing trout with moderate ventricular hypertrophy had lower capillary length densities (1103±58 mm mm⁻³) and similar diffusion distances (13.9±0.7 μm) compared with immature fish (11.7±0.9 μm). The largest ventricles had intermediate capillary length densities (1457±288 mm mm⁻³) and diffusion distances (12.8±0.8 μm). Modelling predicted that enlarged ventricles would not become anoxic even at maximal workloads. Biochemical markers of fatty acid metabolism and aerobic capacity were unchanged with hypertrophy. Volume densities of mitochondria and myofibrils were also not influenced by cardiac growth. In summary, ventricle hypertrophy results in expansion of the coronary capillary bed and the maintenance of the epicardial capacities for fat and oxidative metabolism.     

Pyruvate carboxylase from rainbow trout liver

Summary A method is described for the partial purification of pyruvate carboxylase from rainbow trout liver. The enzyme has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K⁺ causes a decrease in the apparentK _m for HCO ₃ ^– . AcetylCoA activation shows positive cooperativity withK _a=0.072 mM andn _H=1.78 at pH 7.7, 2.5 mM free Mg²⁺, 100 mM K⁺, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparentK _m values for MgATP and HCO ₃ ^– and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a U-type response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control the rate of pyruvate carboxylation in vivo.Abbreviations DTT dithiothreitol - PMSF phenylmethylsulfonylfluoride     

Effects of cortisol on aggression and locomotor activity in rainbow trout

Noninvasive administration of cortisol through the diet resulted in relatively rapid (<1.5 h) and highly reproducible increases in plasma cortisol in rainbow trout, comparable to changes seen in fish subjected to substantial stress. Juvenile rainbow trout were reared in isolation for 1 week, before their daily food ration was replaced by a meal of cortisol-treated food corresponding to 6 mg cortisol kg(-1). All fish were observed for 30 min, beginning at 1 or 48 h following the introduction of cortisol-treated food. Additional cortisol (75% of the original dose on Day 2, and 50% on Day 3) was administered to the long-term cortisol-treated group. The resulting blood plasma concentrations of cortisol were similar in short- and long-term treated fish, and corresponded to those previously seen in stressed rainbow trout. Controls were fed similar food without cortisol. Half of the fish from each treatment group (controls and short- and long-term cortisol) were subjected to an intruder test (a smaller conspecific introduced into the aquarium), while half of the fish were observed in isolation. In fish challenged by a conspecific intruder, short-term cortisol treatment stimulated locomotor activity, while long-term treatment inhibited locomotion. Aggressive behavior was also inhibited by long-term cortisol treatment, but not by short-term exposure to cortisol. Cortisol treatment had no effect on locomotor activity in undisturbed fish, indicating that the behavioral effects of cortisol were mediated through interaction with other signal systems activated during the simulated territorial intrusion test. This study demonstrates for the first time that cortisol has time- and context-dependent effects on behavior in teleost fish.     

Diel variation in branchial calcium uptake by the rainbow trout

Branchial Ca uptake varied dielly with a nadir at 1000 hours and two peaks at 1600 and 0400 hours in rainbow trout. This variation profile essentially reflected Ca uptake through the arterio-arterial circulation.