Cold-acclimated Trout Research Articles

Red muscle function and thermal acclimationto cold in rainbow smelt, Osmerus mordax, and rainbow trout, Oncorhynchus mykiss.

Climate change affects the thermal environment of aquatic organisms. Changes in the thermal environment may affect muscle function in the eurythermal rainbow smelt, Osmerus mordax, and relatively more stenothermal rainbow trout, Oncorhynchus mykiss. Literature suggests that the trout will be more sensitive to changes in environmental temperature, as they experience a more limited range of environmental temperatures. To examine the effects of thermal environment on red muscle function, both the smelt and trout were thermally acclimated to either a warm (12-15°C) or cold (4-5°C) temperature, after which studies of swimming performance and muscle mechanics were performed. The data on swimming performance and maximum muscle shortening velocity in rainbow smelt were previously published. In both species, cold-acclimated (CA) fish swam with a significantly faster maximum aerobic swimming speed than warm-acclimated fish, when tested at a common temperature of 10°C. Similarly, CA smelt and trout had faster red muscle contraction kinetics. However, smelt displayed a greater shift in contractile properties, such as having a significant shift in maximum muscle shortening velocity that was not observed in trout. The smelt red muscle outperformed trout, with twitch and tetanic times of relaxation being significantly faster for CA smelt compared with CA trout, especially when contraction kinetics were tested at 2°C. The smelt shows a greater thermal acclimation response compared with trout, with more robust increases in maximum swimming speed and faster muscle contractile properties. These differences in acclimation response may contribute to understanding how smelt and trout cope with climate change.

An affinity for biochemical adaptation to temperature.

![Figure][1] Alex Gunderson and Jonathon Stillman discuss Peter Hochachka and George Somero's classic paper ‘The adaptation of enzymes to temperature’, published in Comparative Biochemistry and Physiology in 1968. If you have had the pleasure of diving under sea ice in Antarctica, you

Species- and chamber-specific responses of 12 kDa FK506-binding protein to temperature in fish heart

The sarcoplasmic reticulum (SR) Ca(2+) release channel or ryanodine receptor (RyR) of the vertebrate heart is regulated by the FK506-binding proteins, FKBP12 and FKBP12.6. This study examines whether temperature-related changes in the SR function of fish hearts are associated with changes in FKBP12 expression. For this purpose, a polyclonal antibody against trout FKBP12 was used to compare FKPB12 expression in cold-acclimated (4°C, CA) and warm-acclimated (18°C, WA) rainbow trout (Oncorhynchus mykiss), burbot (Lota lota) and crucian carp (Carassius carassius) hearts. FKBP12 expression was modulated in a species- and tissue-specific manner. Temperature acclimation affected FKBP12 expression only in atrial tissue. Changes in the ventricular FKBP12 expression were not detected in any of the fish species. In the atria of rainbow trout and crucian carp, temperature acclimation produced opposite thermal responses: FKBP12 increased in the trout atrium and decreased in the crucian carp atrium under cold acclimation. In the burbot heart, chronic temperature changes did not affect cardiac FKBP12 levels. Expression of FKBP12 mRNA in rainbow trout and crucian carp hearts suggests that the transcript levels are higher in the ventricle than in the atrium and are elevated by cold acclimation in trout, but not in crucian carp. Since FKBP12 is known to increase the Ca(2+) sensitivity of cardiac RyRs and thereby the opening frequency of the Ca(2+) release channels, temperature-related changes in FKBP12 expression may modify the SR function in excitation-contraction coupling. The cold-induced increase in FKBP12 in the trout atrium and decrease in the crucian carp atrium are consistent with the previously noted increase and decrease, respectively, of SR Ca(2+) stores in cardiac contraction in these species.

Expression of SERCA and phospholamban in rainbow trout (Oncorhynchus mykiss) heart: comparison of atrial and ventricular tissue and effects of thermal acclimation

In the heart of rainbow trout (Oncorhynchus mykiss), the rate of contraction and Ca(2+) uptake into the sarcoplasmic reticulum (SR) are faster in atrial than ventricular muscle, and contraction force relies more on SR Ca(2+) stores after acclimation to cold. This study tested the hypothesis that differences in contractile properties and Ca(2+) regulation between atrial and ventricular muscle, and between warm-(WA) and cold-acclimated (CA) trout hearts, are associated with differences in expression of sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) and/or phospholamban (PLN), an inhibitor of the cardiac SERCA. Quantitative PCR (SERCA only) and antibodies raised against SERCA and PLN were used to determine abundances of SERCA2 transcripts and SERCA and PLN proteins, respectively, in atrium and ventricle of trout acclimated to cold (+4°C, CA) and warm (+18°C, WA) temperatures. Expression of SERCA2 transcripts was 1.6 and 2.1 times higher in atrium than ventricle of WA and CA trout, respectively (P<0.05). At the protein level, differences in SERCA expression between atrium and ventricle were 6.1- and 23-fold for WA and CA trout, respectively (P<0.001). Acclimation to cold increased SERCA2 transcripts 2.6- and 2.0-fold in atrial and ventricular muscle, respectively (P<0.05). At the protein level, cold-induced elevation of SERCA (4.6-fold) was noted only in atrial (P<0.05) but not in ventricular tissue (P>0.05). The expression pattern of PLN was similar to that of the SERCA protein, but chamber-specific and temperature-induced differences were much smaller than in the case of SERCA. In the ventricle, PLN/SERCA ratio was 2.1 and 7.0 times higher than in the atrium for WA and CA fish, respectively. These findings are consistent with the hypothesis that low PLN/SERCA ratio in atrial tissue enables faster SR Ca(2+) reuptake and thus contributes to faster kinetics of contraction in comparison with ventricular muscle. Similarly, cold-induced decrease in PLN/SERCA ratio may be associated with faster contraction kinetics of the CA trout heart, in particular in the atrial muscle.

Electrophysiological properties and expression of the delayed rectifier potassium (ERG) channels in the heart of thermally acclimated rainbow trout

In ectotherms, compensatory changes in ion channel number and activity are needed to maintain proper cardiac function at variable temperatures. The rapid component of the delayed rectifier K+ current (IKr) is important for repolarization of cardiac action potential and, therefore, crucial for regulation of cellular excitability and heart rate. To examine temperature plasticity of cardiac IKr, we cloned the ether-à-go-go-related gene (ERG) channel and measured its electrophysiological properties in thermally acclimated rainbow trout (Oncorhynchus mykiss; omERG). The present findings demonstrate a complete thermal compensation in the whole cell conductance of the atrial IKr in rainbow trout acclimated to 4 degrees C (cold acclimation) and 18 degrees C (warm acclimation). In situ hybridization indicates that transcripts of the omERG channel are present throughout the muscular tissue of the heart, and quantitative PCR shows increased expression of the omERG in cold-acclimated trout compared with warm-acclimated trout. In both acclimation groups, omERG expression is higher in atrium than ventricle. In addition, the omERG has some functional features that support IKr activity at low temperatures. Voltage dependence of steady-state activation is completely resistant to temperature changes, and steady-state inactivation and activation kinetics are little affected by temperatures below 11 degrees C. Collectively, these findings suggest that high density of cardiac IKr is achieved by cold-induced increase in the number of functional omERG channels and inherent insensitivity of the omERG to temperature below 11 degrees C. These adaptations are probably important in maintaining high heart rates and proper excitability and contractility of trout cardiac myocytes in the cold.

BEATING THE COLD

Ectothermic animals don't thrive in the cold: low temperature slows down the heart rate, which tends to decrease its output and makes animals slow and sluggish. However, some fish maintain an active lifestyle in the cold. Upon prolonged exposure to low temperature, these fish recruit compensatory mechanisms that bypass the depressive effect of cold temperature on heart rate, enabling the heart to continue to beat at a high rate and precluding an active lifestyle.The fish heart beats independent of external stimulation due to the tightly regulated firing of action potentials (APs) from a specialized set of heart muscle cells called the pacemaker. This regular firing results from coordinated ion movements in the muscle cells. The cold-induced, compensatory increase in heart rate could be due to alterations in humoral and neural regulation of cardiac pacemaker activity and/or modification of the ionic currents underlying the regular AP firing in the pacemaker.Jaako Haverinen and Matti Vornanen of the University of Joensuu, Finland,were interested in determining if and how thermal compensation of the rainbow trout (Oncorhynchus mykiss) heart occurs at the level of the primary pacemaker cells. Because the exact location of the pacemaker cells in the fish heart is not known, the researchers first located the pacemaker region of the fish heart by systematically impaling spontaneously contracting fish hearts with sharp microelectrodes to record APs. These have a specific shape depending on which heart region they are recorded from. Using this technique,and examining the heart tissue, the team discovered that the primary pacemaker in the rainbow trout heart is located in a small ring of tissue between the sinus venosus, the first chamber of a fish's heart, and the atrium.Armed with this knowledge, the team then enzymatically isolated individual pacemaker cells to find out if cold compensation occurred at the cellular level. Using patch-clamping, the team recorded APs from isolated pacemaker cells from both 4°C (cold)- and 18°C (warm)-acclimated fish at the common temperature of 11°C and found that the duration of pacemaker APs was shorter and the intrinsic pace-making rate higher in cold-compared to warm-acclimated trout, showing that at least part of the compensatory increase in heart rate is inherent to the pacemaker cells.Finally, to understand which ionic mechanisms were responsible for the cold-induced changes in pacemaker AP shape and frequency, the team compared APs recorded from spontaneously contracting pacemaker tissue preparations from warm- and cold-acclimated fish. They added chemical blockers specific to two systems: sarcoplasmic reticulum calcium ion (Ca2+) cycling and the movement of potassium ions (K+) through channels called delayed rectifier K+ channels. Both of these ionic mechanisms are implicated as being important for cardiac pacemaking in mammals and are enhanced in cold-acclimated trout.The team found that blocking sarcoplasmic reticulum Ca2+ cycling did not modify pacemaker AP shape or frequency in cold-acclimated tissue. This, they argue, eliminates the possibility that this mechanism underlies the compensatory increase of heart rate. By contrast, the team discovered that the movement of K+ increased in the cold and could be important for increasing heart rate. When they blocked the K+ channel at the common temperature of 11°C, pacemaker AP frequency and duration decreased more in warm-acclimated preparations than in cold-acclimated ones. The team explains that the greater flow of K+ in pacemaker cells of cold-acclimated trout should theoretically shorten AP duration, increase AP discharge frequency and thus increase heart rate.

Temperature acclimation modifies sinoatrial pacemaker mechanism of the rainbow trout heart

The hypothesis of pacemaker level origin of thermal compensation in heart rate was tested by recording action potentials (AP) in intact sinoatrial tissue and enzymatically isolated pacemaker cells of rainbow trout acclimated at 4 degrees C (cold) and 18 degrees C (warm). With electrophysiological recordings, the primary pacemaker was located at the base of the sinoatrial valve, where a morphologically distinct ring of tissue comprising myocytes and neural elements was found by histological examination. Intrinsic beating rate of this pacemaker was higher in cold-acclimated (46 +/- 6 APs/min) than warm-acclimated trout (38 +/- 3 APs/min; P < 0.05), and a similar difference was seen in beating rate of isolated pacemaker cells (44 +/- 6 vs. 38 +/- 6 APs/min; P < 0.05), supporting the hypothesis that thermal acclimation modifies the intrinsic pacemaker mechanism of fish heart. Inhibition of sarcoplasmic reticulum (SR) with 10 microM ryanodine and 1 microM thapsigargin did not affect heart rate in either warm- or cold-acclimated trout at 11 degrees C but reduced heart rate in warm-acclimated trout from 74 +/- 2 to 42 +/- 6 APs/min (P < 0.05) at 18 degrees C. At 11 degrees C, a half-maximal blockade of the delayed rectifier K+ current (I(Kr)) with 0.1 microM E-4031 reduced heart rate more in warm-acclimated (from 45 +/- 1 to 24 +/- 5 APs/min) than cold-acclimated trout (56 +/- 3 vs. 48 +/- 2 APs/min), whereas I(Kr) density was higher and AP duration less in cold-acclimated trout (P > 0.05). Collectively, these findings suggest that a cold-induced increase in AP discharge frequency is at least partly due to higher density of the I(Kr) in the cold-acclimated trout, whereas contribution of SR Ca2+ release to thermal compensation of heart rate is negligible.

Thermally induced changes in lipid composition of raft and non-raft regions of hepatocyte plasma membranes of rainbow trout

In poikilotherms, increases in plasma membrane (PM) cholesterol and an increase in the degree of lipid acyl chain saturation commonly accompany an increase in growth temperature. This has typically been interpreted in terms of membrane fluidity/order homeostasis, but these changes would also be expected to stabilize the structure of PM rafts against thermal perturbation. Rafts are microdomains that organize the molecules of many signaling cascades and are formed as a result of interactions between lipids with saturated acyl chains and cholesterol. No study to date has examined the thermally induced compositional changes of raft and non-raft regions of the PM separately. In this study we have measured the phospholipid class composition and fatty acid composition of raft-enriched (raft) and raft-depleted PM (RDPM) of hepatocytes from trout Oncorhynchus mykiss acclimated to 5 degrees C and 20 degrees C. In the raft, warm acclimation was associated with a reduction in the proportion of phosphatidylcholine from 56% to 30% while phosphatidylserine and phosphatidylinositol each increased from 8% to approximately 20% of the total phospholipid. Additionally, there were significantly fewer unsaturated fatty acids in the raft lipids from warm-acclimated (61%) than from the cold-acclimated trout (68%). In contrast, there were no significant changes in phospholipid class or acyl chain unsaturation in the RDPM. These data suggest that changes in raft lipid composition, rather than the PM as a whole, are particularly important during thermal acclimation.

Steady-state effects of temperature acclimation on the transcriptome of the rainbow trout heart

Cold-acclimated (CA) phenotype of trout heart was induced by 4-wk acclimation at 4 degrees C and was characterized by 32.7% increase in relative heart mass and 49.8% increase in ventricular myocyte size compared with warm-acclimated (WA; 18 degrees C) fish (P < 0.001). Effect of temperature acclimation on transcriptome of the rainbow trout heart was examined using species-specific microarray chips containing 1,380 genes. After 4 wk of temperature acclimation, 8.8% (122) of the genes were differently expressed in CA and WA hearts, and most of them (82%) were upregulated in the cold (P < 0.01). Transcripts of genes engaged in protein synthesis and intermediary metabolism were most strongly upregulated, whereas genes contributing to the connective tissue matrix were clearly repressed. Extensive upregulation of the genes coding for ribosomal proteins and translation elongation and initiation factors suggest that the protein synthesis machinery of the trout heart is enhanced in the cold and is an essential part of the compensatory mechanism causing and maintaining the hypertrophy of cardiac myocytes. The prominent depression of collagen genes may be indicative of a reduced contribution of extracellular matrix to the remodeling of the CA fish heart. Temperature-related changes in transcripts of metabolic enzymes suggest that at mRNA level, glycolytic energy production from carbohydrates is compensated in the heart of CA rainbow trout, while metabolic compensation is absent in mitochondria. In addition, the analysis revealed three candidate genes: muscle LIM protein, atrial natriuretic peptide B, and myosin light chain 2, which might be central for induction and maintenance of the hypertrophic phenotype of the CA trout heart. These findings indicate that extensive modification of gene expression is needed to maintain the temperature-specific phenotype of the fish heart.

Membrane order conservation in raft and non-raft regions of hepatocyte plasma membranes from thermally acclimated rainbow trout

Homeoviscous adaptation (HVA), the thermal conservation of membrane fluidity/order at different body temperatures, has been observed to varying degrees in different membranes. However, HVA has not been studied in raft and non-raft regions of the plasma membrane (PM) separately. Rafts are ordered PM microdomains implicated in signal transduction, membrane traffic and cholesterol homeostasis. Using infrared spectroscopy, we measured order in raft-enriched PM (raft) and raft-depleted PM (RDPM) isolated from hepatocytes of rainbow trout ( Oncorhynchus mykiss) acclimated to 5 and 20 °C. We found approximately 130% and 90% order compensation in raft and RDPM, respectively, suggesting their independent regulation. Raft was more ordered than RDPM in the warm-acclimated trout, a difference fully explained by a 58% enrichment of cholesterol, compared to RPDM. Unexpectedly, raft and RDPM from cold-acclimated trout did not differ in cholesterol content or order. Freezing the membrane samples during preparation had no effect on order. Treatment with cyclodextrin depleted cholesterol by 36%, 56%, and 55%, producing significant decreases in order in raft and RDPM from warm-acclimated trout and RDPM from cold-acclimated trout, respectively. However, a 69% depletion of cholesterol from raft from cold-acclimated trout had no significant effect on order. This result, and the lack of a difference in order between raft and RDPM, suggests that raft and non-raft PM in cold-acclimated trout are not spatially segregated by phase separation due to cholesterol.

Effects of adenosine on the contractility of normoxic rainbow trout heart

Temperature strongly affects oxygen solubility in water, oxygen convection in the blood and locomotor activity of the fish. Since oxygen supply and demand are temperature dependent, it was hypothesized that the purinergic control of the heart, one of the most important mediators in oxygen-limited conditions, might also show temperature dependence. Therefore, the present study examines the effects of adenosine (Ado), a purinergic agonist, on the contractile and electrical activity of the thermally acclimated trout ( Oncorhynchus mykiss Walbaum) heart. The fish were acclimated to either 4 degrees C or 17 degrees C and the experiments were conducted at the acclimation temperatures of the animals. In spontaneously beating hearts, Ado had a negative chronotropic and a positive inotropic effect in warm-acclimated rainbow trout while no response was detected in cold-acclimated trout. In paced atrial and ventricular preparations, Ado had a negative inotropic effect in both warm- and cold-acclimated fish, and the response was strongest in the atria of warm-acclimated trout. Ado shortened the duration of contraction 12-14% in atrial preparations but had no effect in ventricular muscle. Ado (10(-4) mol l(-1)) increased the density of the inwardly rectifying K(+) current from -3.5+/-0.6 pA pF(-1) to -8.4+/-1.4 pA pF(-1) (at -120 mV) in atrial myocytes of warm-acclimated trout but was without effect in atrial myocytes of cold-acclimated trout (-2.4+/-0.8 pA pF(-1) vs. -2.1+/-0.9 pA pF(-1)). Ado had no effect on K(+) currents of ventricular cells in either acclimation group. These results indicate that the effects of Ado on cardiac contractility and electrical activity are stronger in warm-acclimated than in cold-acclimated trout when measured at the physiological body temperatures of the fish. The balance between oxygen demand and supply of the heart might be better in the cold where more environmental oxygen is available and the power of the muscles is weaker thereby reducing the need for the purinergic control of the heart. Temperature-dependence of Ado response in the trout heart warrants that temperature should be taken into consideration when the purinergic system of the ectotherms is studied.

Cold acclimation increases basal heart rate but decreases its thermal tolerance in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss, Walbaum) were acclimated to 4 degrees C and 17 degrees C for more than 4 weeks and heart rate was determined in the absence and presence of adrenaline to see how thermal adaptation influences basal heart rate and its beta-adrenergic control in a eurythermal fish species. The basal heart rate in vitro was higher in cold-acclimated than warm-acclimated rainbow trout at temperatures below 17 degrees C. On the other hand, adaptation to cold decreased thermal tolerance of heart rate so that the maximal heart rates were achieved at 17 degrees C (75 +/- 4 bpm) and 24 degrees C (88 +/- 2 bpm) in cold-acclimated and warm-acclimated trout, respectively. Beta-adrenergic response of the heart was enhanced by cold-adaptation, since adrenaline (100 nmol l(-1)) caused stronger stimulation of heart rate in cold-acclimated (29 +/- 14%) than in warm-acclimated fish (10 +/- 1%; P = 0.03). Furthermore, adrenaline strongly opposed the temperature-dependent deterioration of force production in cold-acclimated trout but not in warm-acclimated trout. The results indicate that adaptation to cold increases basal heart rate but decreases its thermal tolerance in rainbow trout. Cold acclimation up-regulates the beta-adrenergic system, and beta-adrenoceptor activation seems to provide cardioprotection against high temperatures in the cold-adapted rainbow trout.

A respirometric analysis of fuel use during aerobic swimming at different temperatures in rainbow trout (Oncorhynchus mykiss)

Instantaneous fuel usage at 5 degreesC or 15 degreesC was assessed by measurement of rates of O2 consumption (O2), CO2 excretion (CO2) and nitrogenous waste excretion (nitrogen =ammonia-N + urea-N) in juvenile rainbow trout (Oncorhynchus mykiss) at rest and during swimming at 45 % and 75 % of aerobic capacity (Ucrit). After 2 weeks of training at approximately 1 body length s-1 (BL s-1), critical swimming speeds (approximately 3.0 BL s-1) and whole-body energy stores (total protein, lipids and carbohydrates) were identical in fish acclimated to 5 degreesC or 15 degreesC. O2 and CO2 increased with swimming speed at both temperatures and were higher at 15 degreesC than at 5 degreesC at all speeds, but the overall Q10 values (1.23-1.48) were low in these long-term (6 weeks) acclimated fish. The respiratory quotient (CO2/O2, approximately 0.85) was independent of both temperature and swimming speed. In contrast to O2 and CO2, the rate of ammonia excretion was independent of swimming speed, but more strongly influenced by temperature (Q10 1. 4-2.8). Urea excretion accounted for 15-20 % of nitrogen, was unaffected by swimming speed and showed a tendency (P<0.07) to be positively influenced by temperature at one speed only (45 % Ucrit). Nitrogen quotients (NQ nitrogen/O2) were generally higher in warm-acclimated fish, remaining independent of swimming speed at 15 degreesC (0.08), but decreased from about 0.08 at rest to 0.04 during swimming at 5 degreesC. Instantaneous aerobic fuel use calculations based on standard respirometric theory showed that both acclimation temperature and swimming speed markedly influenced the relative and absolute use of carbohydrates, lipids and proteins by trout. At rest, cold-acclimated trout used similar proportions of carbohydrates and lipids and only 27 % protein. During swimming, protein use decreased to 15 % at both speeds while the relative contributions of both lipid and carbohydrate increased (to more than 40 %). On an absolute basis, carbohydrate was the most important fuel for fish swimming at 5 degreesC. In contrast, resting fish acclimated to 15 degreesC utilized 55 % lipid, 30 % protein and only 15 % carbohydrate. However, as swimming speed increased, the relative contribution of carbohydrate increased to 25 %, while the protein contribution remained unchanged at approximately 30 %, and lipid use decreased slightly (to 45 %). On an absolute basis, lipid remained the most important fuel in fish swimming at 15 degreesC. These results support the concept that lipids are a major fuel of aerobic exercise in fish, but demonstrate that the contribution of protein oxidation is much smaller than commonly believed, while that of carbohydrate oxidation is much larger, especially at higher swimming speeds and colder temperature.

Thermal acclimation of phase behavior in plasma membrane lipids of rainbow trout hepatocytes.

The fluorescent probes laurdan (6-dodecanoyl-2-dimethylaminonapthalene) and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl] dipalmitoyl-L-alpha-phosphatidylethanolamine (NBD-PE) in addition to Fourier transform infrared spectroscopy (FTIR) were employed to measure the phase behavior and physical properties of hepatocyte plasma membranes isolated from the livers of thermally acclimated (5 and 20 degreesC) rainbow trout (Oncorhynchus mykiss). The primary objective was to determine the extent to which the phase behavior of membrane lipids is conserved at different growth temperatures. Arrhenius plots of laurdan-generalized polarization revealed a single discontinuity believed to reflect either the onset of the gel-fluid phase transition or the formation of gel phase microdomains, and this discontinuity occurred at significantly higher temperatures in membranes of 20 degrees C (13.2 +/- 0.7 degrees C)- than 5 degrees C (7.2 +/- 0.1 degrees C)-acclimated trout. Similarly, acclimation from 5 to 20 degrees C increased both the onset temperature (from 2.0 +/- 0.3 to 7.2 +/- 0.6 degrees C) and the thermal range (from 10.9 +/- 0.5 to 16.0 +/- 1.0) of the gel-fluid transition as assessed by FTIR. The gel-fluid transition midpoint (approximately -2 degrees C) and completion temperatures (-9 degrees C) were unchanged by thermal acclimation. The anisotropy of NBD-PE fluorescence displayed a distinct minimum in membranes of both warm- and cold-acclimated trout (reflecting alterations in lipid packing that in pure lipid membranes ultimately lead to the formation of nonlamellar phases) in the range of 56-58 degrees C; only membranes of 5 degrees C-acclimated trout displayed an additional minimum at significantly lower temperatures (24.5 +/- 1.7 degrees C). Collectively, these data suggest that the regulation of both the temperature at which gel phase lipids begin to form in response to cooling as well as the propensity of membrane lipids to form nonlamellar phases at higher temperatures may be key features of membrane organization subject to adaptive regulation.

Ca2+-ATPase activity and Ca2+ uptake by sarcoplasmic reticulum in fish heart: effects of thermal acclimation.

This study was designed to compare the activities of sarcoplasmic (SR) Ca2+-ATPase and Ca2+ uptake in fish and mammalian hearts and to determine whether thermal acclimation has any effect on the function of the cardiac SR in fish. To this end, we measured thapsigargin-sensitive Ca2+-ATPase activity and thapsigargin-inhibitable Ca2+ uptake velocity in crude cardiac homogenates of newborn and adult rats and of two teleost fish (crucian carp and rainbow trout) acclimated to low (4 degrees C) and high (17 degrees C and 24 degrees C for trout and carp, respectively) ambient temperatures. The TG-sensitive Ca2+-ATPase activity was highest in adult rat, and the corresponding activities of cold-acclimated trout, warm-acclimated trout, warm-acclimated carp, cold-acclimated carp and newborn rat were 76, 58, 43, 28 and 23 %, respectively, of that of the adult rat at 25 degrees C. SR Ca2+ uptake velocity, measured using Fura-2 at room temperature (approximately 22 degrees C), was highest in cold-acclimated trout, and the values for adult rat, warm-acclimated trout, newborn rat, warm-acclimated carp and cold-acclimated carp were 93, 56, 24, 21 and 14 % of the uptake velocity of cold-acclimated trout, respectively. When corrected to the body temperature of the animal, the relative rates of SR Ca2+ uptake were 100, 26, 19, 18, 11 and 2 % for adult rat, newborn rat, cold-acclimated trout, warm-acclimated trout, warm-acclimated carp and cold-acclimated carp, respectively. These findings show that SR Ca2+ uptake is slower in fish than in mammalian hearts and that marked species-specific differences exist among teleost fish in this respect. Furthermore, acclimation to cold increases the Ca2+ uptake rate of trout cardiac SR (complete thermal compensation) but decreases the SR Ca2+ uptake rate of crucian carp heart. This difference in acclimation response probably reflects the different activity patterns of the two species in their natural habitat during the cold season.

Effects of temperature variations on dietary lipid absorption and plasma lipoprotein concentrations in trout ( Oncorhynchus mykiss)

In juvenile rainbow trout ( Oncorhynchus mykiss) the peak of dietary lipid absorption occurred later at low compared with high temperature. Rapid temperature variations modified plasma concentrations of glucose, lipids and lipoproteins, with no effect on free glycerol. The concentration of these parameters was not affected during the progressive seasonal temperature changes. The plasma concentration of very low density lipoproteins (VLDL) increased by two- to three-fold within two days after the onset of cold acclimation (CA) or warm acclimation, independently of alterations in dietary lipid absorption. The high concentration of VLDL induced an increase in low density lipoproteins (LDL) concentration after several days. CA induced a rapid and transitory decrease in the concentration of high density lipoproteins (HDL) of about 20%. Incomplete successive acclimations resulted in a cumulative effect on the increase of VLDL (10-fold) and of LDL (three-fold). In cold-acclimated trout, the persistent hypertriglyceridemia and high plasma concentration of VLDL observed for several weeks after fasting suggest a long-term increase in the secretion of hepatic VLDL. The results indicated that plasma lipoprotein concentrations appear to be sensitive to the thermal adaptation process in trout exposed to rapid temperature variations.

Qualitative modification of muscle metabolic organization with thermal acclimation of rainbow trout, Oncorhynchus mykiss

Rainbow trout (Oncorhynchus mykiss) were acclimated to 4 and 18°C and fed at rations levels which led to an equal change in mass at these temperatures during the six week acclimation. Thermal acclimation markedly modified the metabolic organization of red and white muscle. Cold-acclimated fish had activities of β-hydroxyacyl CoA dehydrogenase in both red and white muscle that were twice those of warm-acclimated fish. By contrast, the activities of cytochrome oxidase were unchanged by thermal acclimation. Thus, the capacity for β-oxidation of lipids is specifically enhanced in the muscle of cold-acclimated trout. In white muscle, citrate synthase and phosphofructokinase activities were also enhanced by cold acclimation (increases of 125% and 35% respectively), while cytochrome oxidase levels were unchanged. The non-parallel changes in the activities of mitochondrial enzymes strongly suggest that trout muscle mitochondria undergo qualitative reorganization during cold acclimation. The relative activities of mitochondrial enzymes suggest that mitochondria from red muscle have a threefold greater capacity to oxidize lipids than those from white muscle.

The temperature dependence of phospholipid deacylation/reacylation in isolated hepatocytes of thermally acclimated rainbow trout ( Salmo gairdneri)

Rates of glycerol and fatty acid (18:0, 18:1, 18:3 and 20:4) incorporation into phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triacylglycerol were measured in isolated hepatocytes of thermallyacclimated (5° C and 20° C) rainbow trout ( Salmo gairdneri) in order to assess the contribution(s) of the de novo and deacylation/reacylation pathways to the metabolism of membrane lipids. In PC, the effects of acclimation temperature were fatty acid dependent; for 18:0 and 20:4 n − 6 rates of fatty acid incorporation were greater in warm- than cold-acclimated trout, whereas the reverse was true for the incorporation of 18:1. In the case of both PE and triacylglycerol, unsaturated, but not saturated, fatty acids (18:0) were incorporated more rapidly by hepatocytes of cold- than warm-acclimated trout. For PC, ratios of fatty acid / glycerol incorporation were consistently lower than the corresponding ratios for phosphatidic acid regardless of the fatty acid, assay or acclimation temperature. In contrast, fatty acid / glycerol ratios for PE were higher than those for phosphatidic acid in (i) cold- compared to warm-acclimated trout and (ii) in assays conducted at 5°C compared to 20°C for cold-acclimated fish; in addition, ratios generally increased with the degree of unsaturation of the fatty acid in cold-acclimated trout. In the triacylglycerol fraction, fatty acid / glycerol ratios were greater than those for phosphatidic acid in warm-acclimated trout only, and were generally greater in warm- than cold-acclimated fish. These results were interpreted to indicate: (1) an increased contribution of the deacylation/reacylation cycle to the incorporation of unsaturated fatty acids into PE in cold- compared to warm-acclimated fish, and at 5° C compared to 20° C in cold-acclimated trout; and (2) a role for the deacylation/reacylation cycle in excluding the incorporation of unsaturated fatty acids into PC, particularly evident in warm-acclimated trout. The possibility that different metabolic mechanisms are operating to maintain the composition of PE at different temperatures is discussed.

Oxidative and conjugative metabolism of xenobiotics in isolated liver cells from thermally acclimated rainbow trout

Isolated liver cells from rainbow trout were used to study the effects of ambient temperature on xenobiotic biotransformation. The rate-temperature curves show that the cytochrome P-450-dependent metabolism of 7-ethoxycoumarin and benzo( a)pyrene were higher in liver cells from cold-acclimated trout than in warm-acclimated trout at all assay temperatures used. At an incubation temperature close to acclimation temperature (5°C and 15°C, respectively) the rate of metabolism varied very little in liver cells from cold- and warm-acclimated trout, indicating an ideal temperature compensation for these two reactions. In contrast to the cytochrome P-450-dependent metabolism the rate of 7-hydroxycoumarin glucuronidation was significantly lower in liver cells from cold-acclimated trout when compared with warm-acclimated trout and when measured close to the acclimation temperatures, indicating an inversed temperature compensation of the conjugation reaction. The observations in the present study indicate that the ability of rainbow trout to oxidize xenobiotics by cytochrome P-450-dependent metabolism remains constant during acclimation from warm to cold temperature; whereas the ability to conjugate substances with glucuronic acid decreases during the same period. The physiological and toxicological significances of temperature-induced changes on xenobiotic biotransformation enzymes are discussed.

Influence of environmental temperature on the induction of xenobiotic metabolism by β-naphthoflavone in rainbow trout, Salmo gairdneri

The response of hepatic microsomal cytochrome P-450 monooxygenase and UDPglucuronosyltransferase to a common inducer, β-naphthoflavone, was investigated in rainbow trout acclimated to 5 or 17°C. The hepatic microsomal 7-ethoxycoumarin- O-deethylase, 7-ethoxyresorufin- O-deethylase, and benzo[ a]pyrene hydroxylase activities, measured at 5 or 17°C, were induced at both acclimation temperatures. Maximum response of 7-ethoxyresorufin- O-deethylase and 7-ethoxycoumarin- O-deethylase activities to β-naphthoflavone was obtained with doses of 5 mg/kg and above, both in warm- and cold-acclimated trout. In warm-acclimated trout the maximum degree of induction of cytochrome P-450-dependent activities was already evident on the day following treatment with β-naphthoflavone (100 mg/kg), whereas these activities reached maximum values after 3 days in cold-acclimated fish. Furthermore, the maximum induced values of 7-ethoxyresorufin- O-deethylase and benzo[ a]pyrene hydroxylase activities were higher in cold-acclimated than in warm-acclimated fish. β-Napthoflavone treatment caused a slight but significant increase (1.6-fold) in UDPglucuronosyltransferase activity in warm-acclimated fish when measured at 5 or 17°C, whereas in cold-acclimated fish a significant elevation was seen only when the activity was determined at environmental temperature (5°C).