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.     

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.     

Ryanodine and dihydropyridine receptor binding in ventricular cardiac muscle of fish with different temperature preferences

Ca(2+)-induced Ca2+ release (CICR) mechanism of cardiac excitation-contraction (e-c) coupling is dependent on the close apposition between the sarcolemmal dihydropyridine receptors (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptors (RyR). In particular, high RyR/DHPR ratio is considered to reflect strong dependence on SR Ca2+ stores for the intracellular Ca2+ transient. To indirectly evaluate the significance of CICR in fish hearts, densities of cardiac DHPRs and RyRs were compared in ventricular homogenates of three fish species (burbot, rainbow trout, and crucian carp) and adult rat by [3H] PN200-110 and [3H] ryanodine binding. The density of RyRs was significantly (P<0.05) higher in the adult rat (124+/-10 channels/microm3 myocyte volume) than in any of the fish species. Among the fish species, cold-acclimated (4 degrees C) trout had more RyRs than burbot, and crucian carp. The density of DHPRs was highest in the trout heart. RyR/DHPR ratio was significantly (P<0.05) higher in rat (4.1+/-0.5) than in the fish hearts (varying from 0.97+/-0.16 to 1.91+/-0.49) suggesting that "mammalian type" CICR is less important during e-c coupling in fish ventricular myocytes. In rainbow trout, acclimation to cold did not affect the RyR/DHPR ratio, while in crucian carp it was depressed in cold-acclimated animals (4 degrees C; 0.97+/-0.16) when compared to warm-acclimated fish (23 degrees C; 1.91+/-0.49). Although RyR/DHPR ratios were relatively low in fish hearts, there was a close correlation (r2=0.78) between the RyR/DHPR ratio and the magnitude of the Ry-sensitive component of contraction in ventricular muscle among the fish species examined in this study.     

Fatty acid and sterol synthesis by hepatocytes of thermally acclimated rainbow trout (Salmo gairdneri).

Incorporation of tritium from tritiated water into lipid fractions was measured in isolated hepatocytes from rainbow trout (Salmo gairdneri) acclimated to 5 degrees C and 20 degrees C. Hepatocytes from cold-acclimated trout exhibited significantly higher rates of tritium incorporation into both fatty acid and sterol fractions at assay temperatures of 15 degrees C and 20 degrees C than did hepatocytes from warm-acclimated trout. Tritium incorporation into the fatty acid fraction was nearly temperature independent in hepatocytes from warm-acclimated trout (Q10 = 1.39) but markedly temperature dependent (Q10 = 2.63) in hepatocytes from cold-acclimated trout; in contrast, rates of sterol synthesis were more temperature dependent in warm-acclimated trout. At 5 degrees C, fatty acid lipogenesis comprised a significantly greater percentage of the total tritium incorporation in hepatocytes from warm-acclimated trout and the percentage of total lipogenesis attributable to fatty acids decreased significantly in warm-acclimated trout as the assay temperature increased; the opposite trends were observed in cold-acclimated trout.     

Structural studies of natural actomyosin from thermally acclimated frogs.

Natural actomyosin was isolated from skeletal muscle of frogs (Rana catesbeiana) acclimated at 25 degrees C and 5 degrees C. It was found that preparations isolated from warm-acclimated frogs may display considerable degradation of myosin heavy chains as compared with preparations isolated from cold-acclimated frogs. However, degradation may be minimized by inclusion of protease inhibitors during purification, indicating enhanced protease activity in preparations of natural actomyosin from warm-acclimated frogs. When purified in the presence of protease inhibitors, natural actomyosin from both warm-acclimated and cold-acclimated frogs exhibits comparable subunit composition of SDS-gel electrophoresis. The overall gel pattern is similar to that obtained from rabbit natural actomyosin except that in the frog, troponin-T and troponin-C appear to co-migrate with tropomyosin and myosin light chain 2, respectively.     

Role of hyperthyroidism in increased thermogenesis in the cold-acclimated Syrian hamster

The oxygen consumption of cold-acclimated Syrian hamsters (measured in pentobarbital-anaesthetized animals) was 59% greater than that of warm-acclimated hamsters. Upon return of the cold-acclimated hamsters to 24 degrees C, the elevated metabolic rate declined slowly, with half-life of approximately 1 day. The increase correlated well with our previous finding of a four-fold increase in serum 3,5,3'-triiodothyronine concentration in cold-acclimated hamsters and the slow decline in concentration of this hormone during deacclimation. Daily administration of T3 (for 7 days) to warm-acclimated hamsters at 24 degrees C resulted in a similar increase in oxygen consumption that persisted 1 day after the last injection and had disappeared by 7 days after the last injection. We conclude that the high concentration of serum triiodothyronine in the cold-acclimated hamster exerts a thermogenic effect that probably contributes to thermoregulatory thermogenesis in the cold.     

Cardiovascular responses to catecholamines at 12 degrees C in the American bullfrog (Rana catesbeiana)

The effects of epinephrine, norepinephrine, phenylephrine, and isoproterenol on blood pressure and heart rate were studied in cannulated American bullfrogs, Rana catesbeiana. The bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. In warm-acclimated (22 degrees C) bullfrogs, preinjection mean systemic arterial pressure (SAP) prior to experimental treatment was 13.1 +/- 0.7 mm Hg. Preinjection heart rate was 34.8 +/- 1.8 beats per minute. These parameters were lower in cold-acclimated (12 degrees C) bullfrogs. Cold-acclimated animals had mean SAP values of 8.2 +/- 0.3 mm Hg, and heart rate was 11.1 +/- 1.1 beats per minute. Epinephrine, norepinephrine, and phenylephrine increased blood pressure to an equivalent degree in warm- and cold-acclimated animals. Dose-related decreases in heart rate in response to these catecholamines were observed in warm- but not in cold-acclimated bullfrogs. Warm-acclimated animals were more responsive to isoproterenol from 0.03 micrograms/kg body weight (bw) to 10 micrograms/kg bw than were cold-acclimated animals. The response to isoproterenol was effectively blocked by propranolol (5 mg/kg bw) in both warm- and cold-acclimated animals. Propranolol alone decreased mean SAP in both warm- and cold-acclimated animals, suggesting blockade of endogenous sympathetic activity. Beta receptor response thus appears diminished, but not absent at 12 degrees C. However, the alpha receptors responsible for elevation of blood pressure equally responsive at 12 degrees and 22 degrees C.     

Fatty acid synthesis in brown adipose tissue of the Mongolian gerbil (Meriones unguiculatus): influence of acclimation temperature on synthesis in brown adipose tissue and the liver in relation to whole-body synthesis

Fatty acid synthesis was measured in vivo with 3H2O in Mongolian gerbils acclimated at different temperatures. At 31 degrees C the highest rate of synthesis was in the liver, and the liver accounted for approximately one half of whole-body synthesis. At 20 and 40 degrees C, the highest synthesis rate (per g tissue) occurred in brown adipose tissue, and this tissue was estimated to account for some 15% of whole-body synthesis at both these temperatures. In contrast to other rodent species whole-body fatty acid synthesis in the Mongolian gerbil was lower in cold-acclimated than warm-acclimated animals, despite the cold-induced increase in energy intake.     

Characterization of gill Na/K-ATPase activity and ouabain binding in Antarctic and New Zealand nototheniid fishes

The effects of thermal acclimation in two Nototheniid species, the stenothermal Antarctic Trematomous bernacchii and the eurythermal New Zealand Notothenia angustata, were investigated. Serum osmolality, gill Na/K-ATPase activity, sodium pump density and ouabain affinity were determined. Both fish were acclimated at their upper and lower viable thermal temperatures. Warm acclimation (+4 degrees C) of the T. bernacchii significantly decreased their serum osmolality from 550 to 450 mOsm/kg compared to cold-acclimation (-1.5 degrees C) and this was accompanied by a two-fold increase in gill Na/K-ATPase activity. Warm-acclimation (+14 degrees C) of N. angustata did not significantly change their serum osmolality from 330 mOsm/kg or gill Na/K-ATPase activity compared to the cold-acclimated (+4 degrees C) N. angustata. Using [(3)H]ouabain binding techniques, the B(max) and K(d) values of gill Na/K-ATPase enzymes were determined. No difference in the B(max) or K(d) of the warm-acclimated T. bernacchii accounted for the increase in Na/K-ATPase activity. We conclude that the change in gill Na/K-ATPase activity in the warm-acclimated T. bernacchii is not mediated by an increase in the number of enzyme sites and is not reflected in a change in ouabain affinity for Na/K-ATPase.     

Thermal acclimation, growth, and burst swimming of threespine stickleback: enzymatic correlates and influence of photoperiod

Threespine sticklebacks (Gasterosteus aculeatus) that had been reared in the laboratory under natural photoperiods were acclimated to 23 degrees and 8 degrees C in late spring under increasing day lengths and again in late fall under decreasing day lengths. The parents of these fish were from the anadromous Isle Verte population. In the spring, cold- and warm-acclimated fish grew at the same rates and attained similar condition factors (mass L(-3)), although food intake was considerably higher at 23 degrees C. As both groups had similar increases in mass and condition, the higher axial muscle activities of citrate synthase and phosphofructokinase (measured at 20 degrees C) after cold acclimation were likely a direct response to temperature. Multiple regression analysis showed that axial muscle levels of cytochrome C oxidase and citrate synthase were correlated with the burst swimming speeds of the spring sticklebacks, while growth rates were positively correlated with lactate dehydrogenase levels in pectoral and axial muscles and creatine kinase levels in the axial muscle. In the fall, the fish in both acclimation groups grew little, although they fed at similar rates as in the spring experiment. Overall, the sticklebacks showed lower burst swimming speeds in the fall. In both spring and fall, the burst speeds of cold- and warm-acclimated sticklebacks only differed at warm temperatures. In the spring experiment, the cold-acclimated fish swam faster, whereas in the fall experiment the warm-acclimated fish swam faster despite their lower percentage of axial muscle. Swimming speeds were measured both at a fish's acclimation temperature and after 12 h at the other temperature. Cold-acclimated sticklebacks seem to have more facility in rapidly adjusting to warm temperatures when they have experienced increasing rather than decreasing day lengths, perhaps as a result of the requirements of the spring migration to the intertidal breeding grounds.     

Effect of different environmental temperatures on the serotonin concentration and turnover in the brain stem of a hibernator.

The serotonin (5-HT) and 5-hydroxyindoleacttic acid (5-HIAA) levels and 5-HT turnover were studies in the brain stem of warm- (+30 degrees C) and cold- (+6 degrees C) acclimated golden hamsters, exposed for 3 hours to temperatures of +6 degrees C, +30 degrees C and +37 degrees C, respectively. In war-acclimated hamsters kept under conditions the 5-HT level in the brain did not change significantly during the year. The 5-HIAA level was slightly higher in the winter. The 5-HT turnover varied within limits of 0.071 to 0.180 mug/g/hour-1. Three hours' exposure of warm-acclimated golden hamsters to cold (6 degrees C) increased the concentrations of 5-HT and 5-HIAA and the 5-HT turnover in the brain. After long-term adaptation to cold (6 degrees C) the 5-HT level, and the 5-HT turnover returned to the original level. Three hours' exposure of golden hamsters to higher environmental temperatures (warm-acclimated individuals to 37 degrees C and cold-acclimated individuals to 30 degrees C) also increased the 5-HT turnover. The concentrations of 5-HT and 5-HIAA increased in cold-acclimated golden hamsters exposed to 30 degrees C and was not changed in warm-acclimated ones, exposed to 37 degrees C. Although the elevated temperatures induce greater changes in serotonin metabolism than lowered temperatures, the serotonin pathways in the brain do not seem to be affected by short-term temperature changes specifically. The findings are rather indicative that changes in 5-HT turnover may be the primary reaction to stressful conditions.     

Effect of extracellular calcium on the contractility of warm-and cold-acclimated crucian carp heart

Crucian carp (Carassius carassius L.) were acclimated for at least 4 weeks to 2°C or 22°C, and the consequences of thermal acclimation on force development, time-course of contraction and action potential duration of the ventricular myocardium were studied. In cold-acclimated fish contraction was activated at much lower external [Ca] than in warm-acclimated fish: [Ca] for half-maximal force was 0.9±0.15 and 3.1±0.92 mmol·l^-1 (P<0.05) for cold- and warm-acclimated fish, respectively. Durations of contraction and relaxation were significantly longer in fish acclimated to 2°C than in fish acclimated to 22°C, especially at [Ca] below 2 mmol·l^-1. In low-Ca solution ventricular action potential was prolonged both in cold- and warm-acclimated fish. In 0.5 mmol·l^-1 Ca action potential duration at zero voltage level was longer in cold- than warm-acclimated fish. Although lengthening of action potential was evident in both acclimation groups, a marked prolongation of contraction duration by low-Ca solutions occurred only in cold-acclimated fish. This suggests that a plateau component of contraction is present in cold-acclimated fish but less well developed in warm-acclimated fish hearts. Contractions were strongly inhibited by sarcolemmal Ca-channel blocker, cadmium (100 and 300 mol·l^-1), in both warm- and cold-acclimated crucian carp hearts. However, the sarcoplasmic reticulum Ca release channel blocker, ryanodine (10 mol·l^-1), had no effect on the force of contraction in either acclimation group. These results suggest that the contraction of crucian carp heart is controlled by sarcolemmal mechanisms without contribution by sarcoplasmic reticulum Ca release. Since the Ca sensitivity of myofilaments was not altered by thermal acclimation, the results indicate that thermal acclimation alters Ca activation of contraction of the crucian carp heart at the level of sarcolemma.Abbreviations AP action potential - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra-acetic acid - F _max maximum force - F _max maximum rate of contraction - F _min maximum rate of relaxation - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - pCa log [Ca] - Pl action potential plateau - SL sarcolemma - SR sarcoplasmic reticulum - TPF time to peak force - T1/2R time to half relaxation from the peak force     

Temperature dependence and acclimatory response of amylase in the High Arctic springtail Onychiurus arcticus (Tullberg) compared with the temperate species Protaphorura armata (Tullberg)

Amylolytic activity was measured in whole body homogenates of High Arctic (Onychiurus arcticus) and temperate (Protaphorura armata) springtails (Collembola: Onychiuridae) in the temperature range 5-55 degrees C. A pH of ca. 8 was optimum for amylolytic activity in both species. A higher weight-specific amylolytic activity was observed in P. armata. In O. arcticus, amylolytic activity depended on thermal acclimation, which increased during 2 and 9 weeks of cold acclimation (5 degrees C) and decreased over 7 weeks of warming (15 degrees C) of animals that were previously acclimated to cold for 2 weeks. In cold-acclimated O. arcticus, a slower decrease of amylolytic activity occurred with lowering of temperature in the range 5-20 degrees C in comparison with warm-acclimated specimens and P. armata, which resulted in higher activity at 5 degrees C. The activation energy calculated from an Arrhenius plot for P. armata was 68.7 kJ.mol(-1). In O. arcticus it was between 30.2 and 61.5 kJ.mol(-1), being lower in cold-acclimated samples. The temperature optimum for amylolytic activity was higher in the temperate species (40 degrees C), whilst in O. arcticus it depended on the acclimation regime: it rose to 35 degrees C after warm acclimation and decreased to 20 degrees C after cold adaptation. The total soluble protein content of body tissues of O. arcticus also increased during cold acclimation. These differences between the two species suggest that amylolytic activity is an indicator of cold adaptation in the High Arctic O. arcticus.     

Alterations to the swimming performance of carp, Cyprinus carpio, as a result of temperature acclimation

Groups of common carp were acclimated to either 10°C or 28°C for 6 weeks. Fish were then exercised at 10°C or 20°C, and the critical swimming speed (fatigue velocity) was measured. At 10°C, cold-acclimated carp were capable of significantly higher swimming speeds. When exercised at 20°C. however, the situation was reversed, and warm-acclimated carp exhibited improved swimming ability. These results provide direct evidence that acclimation of the contractile proteins is beneficial across a wide temperature range. Following acclimation to low environmental temperatures the critical swimming speed exhibited a Q₁₀ of only 1.1 for the temperature range 10–20°C. compared to a value of 2.9 for fish acclimated to the higher temperature.     

Role of mitochondrial and sarcolemmal K(ATP) channels in ischemic preconditioning of the canine heart

We tested whether mitochondrial or sarcolemmal ATP-sensitive K(+) (K(ATP)) channels play a key role in ischemic preconditioning (IP) in canine hearts. In open-chest beagle dogs, the left anterior descending artery was occluded four times for 5 min each with 5-min intervals of reperfusion (IP), occluded for 90 min, and reperfused for 6 h. IP as well as cromakalim and nicorandil (nonspecific K(ATP) channel openers) markedly limited infarct size (6.3 +/- 1.2, 8.9 +/- 1.9, and 7.2 +/- 1.6%, respectively) compared with the control group (40.9 +/- 4.1%). A selective mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate, partially blunted the limitation of infarct size in the animals subjected to IP and those treated with cromakalim and nicorandil (21.6 +/- 3.8, 25.1 +/- 4.6, and 19.8 +/- 5.2%, respectively). A nonspecific K(ATP) channel blocker, glibenclamide, completely abolished the effect of IP (38.5 +/- 6.2%). Intracoronary or intravenous administration of a mitochondria-selective K(ATP) channel opener, diazoxide, at >100 micromol/l could only partially decrease infarct size (19.5 +/- 4.3 and 20.1 +/- 4.4%, respectively). In conclusion, mitochondrial and sarcolemmal K(ATP) channels independently play an important role in the limitation of infarct size by IP in the canine heart.     

Regulation of action potential duration under acute heat stress by I(K,ATP) and I(K1) in fish cardiac myocytes

The mechanism underlying temperature-dependent shortening of action potential (AP) duration was examined in the fish (Carassius carassius L.) heart ventricle. Acute temperature change from +5 to +18 degrees C (heat stress) shortened AP duration from 2.8 +/- 0.3 to 1.3 +/- 0.1 s in intact ventricles. In 56% (18 of 32) of enzymatically isolated myocytes, heat stress also induced reversible opening of ATP-sensitive K+ channels and increased their single-channel conductance from 37 +/- 12 pS at +8 degrees C to 51 +/- 13 pS at +18 degrees C (Q10 = 1.38) (P < 0.01; n = 12). The ATP-sensitive K+ channels of the crucian carp ventricle were characterized by very low affinity to ATP both at +8 degrees C [concentration of Tris-ATP that produces half-maximal inhibition of the channel (K1/2)= 1.35 mM] and +18 degrees C (K1/2 = 1.85 mM). Although acute heat stress induced ATP-sensitive K+ current (IK,ATP) in patch-clamped myocytes, similar heat stress did not cause any glibenclamide (10 microM)-sensitive changes in AP duration in multicellular ventricular preparations. Examination of APs and K+ currents from the same myocytes by alternate recording under current-clamp and voltage-clamp modes revealed that changes in AP duration were closely correlated with temperature-specific changes in the voltage-dependent rectification of the background inward rectifier K+ current IK1. In approximately 15% of myocytes (4 out of 27), IK,ATP-dependent shortening of AP followed the IK1-induced AP shortening. Thus heat stress-induced shortening of AP duration in crucian carp ventricle is primarily dependent on IK1. IK,ATP is induced only in response to prolonged temperature elevation or perhaps in the presence of additional stressors.