ABSTRACT Tropical lagoon-inhabiting organisms live in highly irradiated ecosystems and are particularly susceptible to thermal stress resulting from climate change. However, despite living close to their thermal maxima, stress response mechanisms found in these organisms are poorly understood. We used a novel physiological–proteomic approach for sponges to describe the stress response mechanisms of the lagoon-inhabiting sponge Amphimedon navalis , when exposed to elevated seawater temperatures of +2°C and +4°C relative to a 26°C ambient temperature for 4 weeks. After 4 weeks of thermal exposure, the buoyant weight of the sponge experienced a significant decline, while its pumping rates and oxygen consumption rates significantly increased. Proteome dynamics revealed 50 differentially abundant proteins in sponges exposed to elevated temperature, suggesting that shifts in the sponge proteome were potential drivers of physiological dysfunction. Thermal stress promoted an increase in detoxification proteins, such as catalase, suggesting that an excess of reactive oxygen species in sponge cells was responsible for the significant increase in oxygen consumption. Elevated temperature also disrupted cellular growth and cell proliferation, promoting the loss of sponge biomass, and the high abundance of multiple α-tubulin chain proteins also indicated an increase in cytoskeletal activities within sponge cells, which may have induced the increase in sponge pumping rate. Our results show that sustained thermal exposure in susceptible lagoonal sponges may induce significant disruption of cellular homeostasis, leading to physiological dysfunction, and that a combined physiological–proteomic approach may provide new insights into physiological functions and cellular processes occurring in sponges.

ABSTRACT A heat stressor (1 h at 30°C) in Lymnaea stagnalis before operant conditioning training of aerial respiration is sufficient to enhance long-term memory (LTM) formation in ‘average’ cognitive ability, laboratory-reared, inbred snails. However, in freshly collected outbred snails, the same heat stressor blocks LTM formation in ‘smart’ cognitive phenotype but not in average cognitive phenotype strains. Here, we hypothesize that (1) preventing the stress associated with the heat stressor before training allows LTM to form in the smart phenotype strains; and (2) alleviating the stress before a memory recall session allows a formed LTM to be recalled in the smart phenotype strains. We found that an injection of propranolol, which mitigates the stressor, before snails experience the heat stressor enabled two strains of the smart phenotype snails to form LTM, consistent with our first hypothesis. However, the injection of propranolol before a memory test session did not alleviate a memory recall block in the smart phenotype snails. Thus, our second hypothesis was not supported. Therefore, smart cognitive phenotype snails encountering a heat stressor have an inability to form LTM, but this inability can be overcome by the pre-injection of propranolol.

ABSTRACT Climate change is increasing the frequency of heat waves and other extreme weather events experienced by organisms. How does the number and developmental timing of heat waves affect survival, growth and development of insects? Do heat waves early in development alter performance later in development? We addressed these questions using experimental heat waves with larvae of the tobacco hornworm, Manduca sexta. The experiments used diurnally fluctuating temperature treatments differing in the number (0–3) and developmental timing (early, middle and/or late in larval development) of heat waves, in which a single heat wave involved three consecutive days with a daily maximum temperature of 42°C. Survival to pupation declined with increasing number of heat waves. Multiple (but not single) heat waves significantly reduced development time and pupal mass; the best models for the data indicated that both the number and developmental timing of heat waves affected performance. In addition, heat waves earlier in development significantly reduced growth and development rates later in larval development. Our results illustrate how the frequency and developmental timing of sublethal heat waves can have important consequences for life history traits in insects.

ABSTRACT Female-biased mortality has been repeatedly reported in Pacific salmon during their upriver migration in both field studies and laboratory holding experiments, especially in the presence of multiple environmental stressors, including thermal stress. Here, we used coho salmon ( Oncorhynchus kisutch ) to test whether females exposed to elevated water temperatures (18°C) (i) suppress circulating sex hormones (testosterone, 11-ketotestosterone and estradiol), owing to elevated cortisol levels, (ii) have higher activities of enzymes supporting anaerobic metabolism (e.g. lactate dehydrogenase, LDH), (iii) have lower activities of enzymes driving oxidative metabolism (e.g. citrate synthase, CS) in skeletal and cardiac muscle, and (iv) have more oxidative stress damage and reduced capacity for antioxidant defense [lower catalase (CAT) activity]. We found no evidence that a higher susceptibility to oxidative stress contributes to female-biased mortality at warm temperatures. We did, however, find that females had significantly lower cardiac LDH and that 18°C significantly reduced plasma levels of testosterone and estradiol, especially in females. We also found that relative gonad size was significantly lower in the 18°C treatment regardless of sex, whereas relative liver size was significantly lower in females held at 18°C. Further, relative spleen size was significantly elevated in the 18°C treatments across both sexes, with larger warm-induced increases in females. Our results suggest that males may better tolerate bouts of cardiac hypoxia at high temperature, and that thermal stress may also disrupt testosterone- and estradiol-mediated protein catabolism, and the immune response (larger spleens), in migratory female salmon.

ABSTRACT Ambient temperature-associated stress can affect normal physiological functions in ectotherms. To assess the effects of cold or heat stress on amphibians, giant spiny frogs ( Quasipaa spinosa ) were acclimated at 22°C followed by exposure to 5°C or 30°C for 0, 3, 6, 12, 24 and 48 h, respectively. Histological alterations, apoptotic index, generation of mitochondrial reactive oxygen species (ROS), antioxidant activity indices and stress-response gene expression in frog livers were subsequently determined. Results showed that many fat droplets appeared after 12 h of heat stress and the percentage of melanomacrophage centres significantly changed after 48 h at both stress conditions. Furthermore, the mitochondrial ROS levels were elevated in a time-dependent manner up to 6 h and 12 h in the cold and heat stress groups, respectively. The activities of superoxide dismutase, glutathione peroxidase and catalase were successively increased with increasing periods of cold or heat exposure, and their gene expression levels showed similar changes in both stress conditions. Most tested heat shock protein (HSP) genes were sensitive to temperature exposure, and the expression profiles of most apoptosis-related genes was significantly upregulated at 3 and 48 h under cold and heat stress, respectively. Apoptotic index at 48 h under cold stress was significantly higher than that under heat stress. Notably, lipid droplets, HSP30 , HSP70 and HSP110 might be suitable bioindicators of heat stress. The results of these alterations at physiological, biochemical and molecular levels might contribute to a better understanding of the stress response of Q. spinosa , and perhaps amphibians more generally, under thermal stress.

ABSTRACT Maternal investment is likely to have direct effects on offspring survival. In oviparous animals whose embryos are exposed to the external environment, maternal provisioning of molecular factors like mRNAs and proteins may help embryos cope with sudden changes in the environment. Here, we sought to modify the maternal mRNA contribution to offspring embryos and test for maternal effects on acute thermal tolerance in early embryos of Drosophila melanogaster . We drove in vivo overexpression of a small heat shock protein gene ( Hsp23 ) in female ovaries and measured the effects of acute thermal stress on offspring embryonic survival and larval development. We report that overexpression of the Hsp23 gene in female ovaries produced offspring embryos with increased thermal tolerance. We also found that brief heat stress in the early embryonic stage (0–1 h old) caused decreased larval performance later in life (5–10 days old), as indexed by pupation height. Maternal overexpression of Hsp23 protected embryos against this heat-induced defect in larval performance. Our data demonstrate that transient products of single genes have large and lasting effects on whole-organism environmental tolerance. Further, our results suggest that maternal effects have a profound impact on offspring survival in the context of thermal variability.

ABSTRACT Preconditioning to non-stressful warming can protect some symbiotic cnidarians against the high temperature-induced collapse of their mutualistic endosymbiosis with photosynthetic dinoflagellates ( Symbiodinium spp.), a process known as bleaching. Here, we sought to determine whether such preconditioning is underpinned by differential regulation of aerobic respiration. We quantified in vivo metabolism and mitochondrial respiratory enzyme activity in the naturally symbiotic sea anemone Exaiptasia pallida preconditioned to 30°C for >7 weeks as well as anemones kept at 26°C. Preconditioning resulted in increased Symbiodinium photosynthetic activity and holobiont (host+symbiont) respiration rates. Biomass-normalised activities of host respiratory enzymes [citrate synthase and the mitochondrial electron transport chain (mETC) complexes I and IV] were higher in preconditioned animals, suggesting that increased holobiont respiration may have been due to host mitochondrial biogenesis and/or enlargement. Subsequent acute heating of preconditioned and ‘thermally naive’ animals to 33°C induced dramatic increases in host mETC complex I and Symbiodinium mETC complex II activities only in thermally naive E. pallida . These changes were not reflected in the activities of other respiratory enzymes. Furthermore, bleaching in preconditioned E. pallida (defined as the significant loss of symbionts) was delayed by several days relative to the thermally naive group. These findings suggest that changes to mitochondrial biogenesis and/or function in symbiotic cnidarians during warm preconditioning might play a protective role during periods of exposure to stressful heating.

Antarctic fish of the suborder Notothenioidei have evolved several unique adaptations to deal with subzero temperatures. However, these adaptations may come with physiological trade-offs, such as an increased susceptibility to oxidative damage. As such, the expected environmental perturbations brought on by global climate change have the potential to significantly increase the level of oxidative stress and cellular damage in these endemic fish. Previous single stressor studies of the notothenioids have shown they possess the capacity to acclimate to increased temperatures, but the cellular-level effects remain largely unknown. Additionally, there is little information on the ability of Antarctic fish to respond to ecologically relevant environmental changes where multiple variables change concomitantly. We have examined the potential synergistic effects that increased temperature and Ṗ CO 2 have on the level of protein damage in Trematomus bernacchii, Pagothenia borchgrevinki and Trematomus newnesi , and combined these measurements with changes in total enzymatic activity of catalase (CAT) and superoxide dismutase (SOD) in order to gauge tissue-specific changes in antioxidant capacity. Our findings indicate that total SOD and CAT activity levels displayed only small changes across treatments and tissues. Short-term acclimation to decreased seawater pH and increased temperature resulted in significant increases in oxidative damage. Surprisingly, despite no significant change in antioxidant capacity, cellular damage returned to near-basal levels, and significantly decreased in T. bernacchii , after long-term acclimation. Overall, these data suggest that notothenioid fish currently maintain the antioxidant capacity necessary to offset predicted future ocean conditions, but it remains unclear whether this capacity comes with physiological trade-offs.

In aquatic systems, physiological processes such as respiration, photosynthesis and calcification are potentially limited by the exchange of dissolved materials between organisms and their environment. The nature and extent of physiological limitation is, therefore, likely to be dependent on environmental conditions. Here, we assessed the metabolic sensitivity of barnacles under a range of water temperatures and velocities, two factors that influence their distribution. Respiration rates increased in response to changes in temperature and flow, with an interaction where flow had less influence on respiration at low temperatures, and a much larger effect at high temperatures. Model analysis suggested that respiration is mass transfer limited under conditions of low velocity (<7.5 cm −1 ) and high temperature (20–25°C). In contrast, limitation by uptake reaction kinetics, when the biotic capacity of barnacles to absorb and process oxygen is slower than its physical delivery by mass transport, prevailed at high flows (40–150 cm s −1 ) and low temperatures (5–15°C). Moreover, there are intermediate flow-temperature conditions where both mass transfer and kinetic limitation are important. Behavioral monitoring revealed that barnacles fully extend their cirral appendages at low flows and display abbreviated ‘testing’ behaviors at high flows, suggesting some form of mechanical limitation. In low flow–high temperature treatments, however, barnacles displayed distinct ‘pumping’ behaviors that may serve to increase ventilation. Our results suggest that in slow-moving waters, respiration may become mass transfer limited as temperatures rise, whereas faster flows may serve to ameliorate the effects of elevated temperatures. Moreover, these results underscore the necessity for approaches that evaluate the combined effects of multiple environmental factors when examining physiological and behavioral performance.

SUMMARY Many endogenous factors influence thermal tolerance of insects. Among these, age contributes an important source of variation. Heat tolerance is typically high in newly eclosed insects, before declining dramatically. It is not known whether this phenomenon relates to cold tolerance also. In addition, the underlying mechanisms of this variation are unresolved. In this study, we tested whether cold tolerance declines in Drosophila melanogaster females aged from 0 to 5 days. We also assessed whether expression (basal and induced) of eight stress genes ( hsp22 , hsp23 , hsp40 , hsp68 , hsp70Aa , hsp83 , Starvin and Frost ) varied post-eclosion in correspondence with changes found in cold tolerance. We report that cold tolerance was very high at eclosion and then it rapidly declined in young flies. hsp23 and hsp68 showed a dramatic age-related variation of basal expression that was associated with cold tolerance proxies. Significant age-related plasticity of cold-induced expression was also found for hsp22 , hsp23 , hsp68 , hsp70Aa , Frost and Starvin. Induced expression of hsp22 and hsp70Aa was high in newly enclosed phenotypes before declining dramatically, whilst opposite age-related patterns were found for hsp23 , hsp68 , Starvin and Frost . This study shows a marked within-stage variation in cold tolerance. The involvement of the stress genes in setting basal thermal tolerance is discussed.

SUMMARY In the present study we investigated the signal transduction cascades triggered by acute thermal stress in Mytilus galloprovincialis gills. This particular species has been reported to exhibit a significant tolerance to high temperatures; thus, it was intriguing to examine the molecular mechanisms responsible for this extraordinary trait. In particular, exposure to 30°C was found to cause a significant and sustained stimulation of p38-MAPK phosphorylation while the activation profile of JNKs was transient and relatively moderate. We also observed that hyperthermia induced apoptosis as a delayed response, with both MAPK subfamilies rapidly translocating to the nucleus. The phosphorylation of cJun, ATF2 and NFκB was detected next. Using selective inhibitors, phosphorylation of these transcription factors was established to be dependent on p38-MAPK or JNKs. Subsequently, potential changes in gene expression were assessed. In this context, hyperthermia resulted in the transcriptional upregulation of Hsp70 and MT20 genes with a widely known salutary effect, preserving mussel fitness and performance under adverse environmental conditions. Interestingly, p38-MAPK and JNKs were found to mediate the hyperthermia-induced Hsp70 and MT20 upregulation as well as the delayed induction of apoptosis under the interventions studied. Overall this is, to our knowledge, the first time that an insight into the compensatory survival `programme' initiated in Mytilus galloprovincialis gills, contributing to this organism's exceptional tolerance to thermal stress, has been gained. In particular, we provide evidence demonstrating the principal role of p38-MAPK and JNKs in transducing the stress signal via mobilization of specific transcription factors and the transcriptional upregulation of cytoprotective genes.

SUMMARY Increase in seawater temperature is one of the major effects of global climate change that affects marine organisms, including Cnidaria. Among them,gorgonians from the NW Mediterranean Sea, such as the species Eunicella singularis , have suffered spectacular and extensive damage. We thus investigated in a controlled laboratory experiment the response of E. singularis to a long-term increase in temperature and we took a special interest in its photosynthetic and calcification response to the stress. Two populations collected at 15 and 35 m depths were studied in order to determine whether there was a difference in sensitivity to thermal stress between living depths. Our results show: (a) that calcification and photosynthesis were impacted only when gorgonians were maintained for more than two weeks at 26°C, and (b) that colonies of E. singularis living in shallow waters were less tolerant than those living in deep waters. Because E. singularis is a symbiotic species, we have also discussed the potential role of symbiosis in the thermotolerance response.

SUMMARY The mutualistic relationship between corals and their unicellular dinoflagellate symbionts ( Symbiodinium sp.) is a fundamental component within the ecology of coral reefs. Thermal stress causes the breakdown of the relationship between corals and their symbionts (bleaching). As with other organisms, this symbiosis may acclimate to changes in the environment, thereby potentially modifying the environmental threshold at which they bleach. While a few studies have examined the acclimation capacity of reef-building corals, our understanding of the underlying mechanism is still in its infancy. The present study focused on the role of recent thermal history in influencing the response of both corals and symbionts to thermal stress, using the reef-building coral Acropora aspera . The symbionts of corals that were exposed to 31°C for 48 h (pre-stress treatment) 1 or 2 weeks prior to a 6-day simulated bleaching event (when corals were exposed to 34°C) were found to have more effective photoprotective mechanisms. These mechanisms included changes in non-photochemical quenching and xanthophyll cycling. These differences in photoprotection were correlated with decreased loss of symbionts, with those corals that were not prestressed performing significantly worse, losing over 40% of their symbionts and having a greater reduction in photosynthetic efficiency. These results are important in that they show that thermal history, in addition to light history, can influence the response of reef-building corals to thermal stress and therefore have implications for the modeling of bleaching events. However, whether acclimation is capable of modifying the thermal threshold of corals sufficiently to cope as sea temperatures increase in response to global warming has not been fully explored. Clearly increases in sea temperatures that extend beyond 1–2°C will exhaust the extent to which acclimation can modify the thermal threshold of corals.

SUMMARY In the present study, we investigated the induction of the p38-MAPK signalling pathway by copper, as exemplified by CuCl 2 , in the isolated perfused heart of the amphibian Rana ridibunda . We found that p38-MAPK phosphorylation by CuCl 2 occurs in a dose-dependent manner, with maximum activation (8.73±1.43-fold relative to control values) attained by perfusion with 500 μmol l –1 CuCl 2 for 15 min, while this activation sustained even after 60 min of reperfusion with normal bicarbonate buffer. CuCl 2 also induced the phosphorylation of the small heat shock protein 27 (Hsp27) in a p38-MAPK dependent manner, as revealed by experiments using the p38-MAPK inhibitor SB203580. p38-MAPK and Hsp27 phosphorylations were also strongly induced by hyperthermia (42°C), while the simultaneous use of hyperthermia and CuCl 2 had a synergistic effect on p38-MAPK activation. Furthermore,perfusions with the potent antioxidant L-ascorbic acid (100 μmol l –1 ), the antioxidant enzymes catalase (CAT) (150 U ml –1 ) or superoxide dismutase (SOD) (30 U ml –1 ) in the presence of 500 μmol l –1 CuCl 2 did not attenuate the CuCl 2 -induced p38-MAPK activation, implying that at least the reactive oxygen species (ROS) scavenged by these agents are not implicated in this kinase activation. The p38-MAPK phosphorylation induced by the combined action of CuCl 2 and hyperthermia was partially inhibited by catalase, indicating that hyperthermia possibly activates the kinase through the production of H 2 O 2 . Caspase-3, an effector protease of apoptosis,remained inactive in hearts perfused at normal or hyperthermic conditions, in the absence or presence of 500 μmol l –1 CuCl 2 . All the above results suggest that, in the amphibian Rana ridibunda heart , p38-MAPK activation by copper has a possible protective role through the small Hsp27.

SUMMARY The shrimp Mirocaris fortunata is a hydrothermal vent species that is found at most vent-sites along the Mid-Atlantic Ridge. This endemic species is found across a hydrothermal gradient, with thermal conditions ranging from 2–9°C in ambient seawater to fairly warm values of about 25°C. We performed in vivo experiments on M. fortunata specimens originating from different sites and depths (850 m to 2300 m), both at atmospheric pressure and in pressurized aquaria, to characterise the upper thermal limits of this species. Atmospheric pressure results show that thermal physiology should be studied at each population's native pressure. At in situ pressure, shrimps from Menez Gwen (850 m depth) and Lucky Strike(1700 m depth) do not survive temperatures of 39°C, and the `loss of equilibrium' response suggests that their critical thermal maximum( Ct max ), is about 36±1°C for both sites. This value is similar to those found for another vent shrimp, Rimicaris exoculata , which is thought to be a more temperature-resistant organism,so temperature resistance does not appear to be a crucial factor for explaining differences in distribution of shrimp species in a given vent site. Finally, the data for both vent shrimps are also comparable to those of other non-vent tropical caridean species.

SUMMARY Cnidarians in symbiosis with photosynthetic protists must withstand daily hyperoxic/anoxic transitions within their host cells. Comparative studies between symbiotic ( Anemonia viridis ) and non-symbiotic ( Actinia schmidti ) sea anemones show striking differences in their response to oxidative stress. First, the basal expression of SOD is very different. Symbiotic animal cells have a higher isoform diversity (number and classes)and a higher activity than the non-symbiotic cells. Second, the symbiotic animal cells of A. viridis also maintain unaltered basal values for cellular damage when exposed to experimental hyperoxia (100% O 2 ) or to experimental thermal stress (elevated temperature +7°C above ambient). Under such conditions, A. schmidti modifies its SOD activity significantly. Electrophoretic patterns diversify, global activities diminish and cell damage biomarkers increase. These data suggest symbiotic cells adapt to stress while non-symbiotic cells remain acutely sensitive. In addition to being toxic, high O 2 partial pressure( P O 2 ) may also constitute a preconditioning step for symbiotic animal cells, leading to an adaptation to the hyperoxic condition and, thus, to oxidative stress. Furthermore, in aposymbiotic animal cells of A. viridis , repression of some animal SOD isoforms is observed. Meanwhile, in cultured symbionts, new activity bands are induced,suggesting that the host might protect its zooxanthellae in hospite . Similar results have been observed in other symbiotic organisms, such as the sea anemone Aiptasia pulchella and the scleractinian coral Stylophora pistillata . Molecular or physical interactions between the two symbiotic partners may explain such variations in SOD activity and might confer oxidative stress tolerance to the animal host.

SUMMARY Photosynthetic endolithic algae and cyanobacteria live within the skeletons of many scleractinians. Under normal conditions, less than 5% of the photosynthetically active radiation (PAR) reaches the green endolithic algae because of the absorbance of light by the endosymbiotic dinoflagellates and the carbonate skeleton. When corals bleach (loose dinoflagellate symbionts),however, the tissue of the corals become highly transparent and photosynthetic microendoliths may be exposed to high levels of both thermal and solar stress. This study explores the consequence of these combined stresses on the phototrophic endoliths inhabiting the skeleton of Montipora monasteriata , growing at Heron Island, on the southern Great Barrier Reef. Endoliths that were exposed to sun after tissue removal were by far more susceptible to thermal photoinhibition and photo-damage than endoliths under coral tissue that contained high concentrations of brown dinoflagellate symbionts. While temperature or light alone did not result in decreased photosynthetic efficiency of the endoliths, combined thermal and solar stress caused a major decrease and delayed recovery. Endoliths protected under intact tissue recovered rapidly and photoacclimated soon after exposure to elevated sea temperatures. Endoliths under naturally occurring bleached tissue of M. monasteriata colonies (bleaching event in March 2004 at Heron Island) acclimated to increased irradiance as the brown symbionts disappeared. We suggest that two major factors determine the outcome of thermal bleaching to the endolith community. The first is the microhabitat and light levels under which a coral grows, and the second is the susceptibility of the coral-dinoflagellates symbiosis to thermal stress. More resistant corals may take longer to bleach allowing endoliths time to acclimate to a new light environment. This in turn may have implications for coral survival.

SUMMARY The critical thermal maxima ( CT max ) of two sympatric,diurnal, thermophilic harvester ants from the Mojave Desert, USA( Pogonomyrmex rugosus and P. californicus ) were measured by ramping their temperature upwards at a rate of 0.25°C min -1 during flow-through respirometry with optical activity detection. Rates of CO 2 emission( V̇ CO 2 ) increased exponentially to plateau values that were twice as high in P. californicus as P. rugosus on a mass-specific basis. V̇ CO 2 then fell sharply, during which gross motor activity (measured optically) and spiracular control (measured from V̇ CO 2 variation)abruptly ceased, yielding two independent measures of CT max . As determined by loss of muscular coordination, the CT max of Pogonomyrmex rugosus was 51.57±0.38°C (mean ± s.d. , while that of Pogonomyrmex californicus was 51.74±0.25°C. As determined by loss of spiracular control, the CT max of Pogonomyrmex rugosus was 51.59±0.35°C, while that of Pogonomyrmex californicus was 51.78±0.37°C. In each species a pronounced post-mortal peak of V̇ CO 2 was observed. The major ecological and behavioral differences of the two species are not reflected in their CT max values, which do not differ significantly. `Thermolimit respirometry' allows CT max to be estimated objectively with coefficients of variation ( s.d. /mean) <1%, lending confidence to comparisons between species or treatment groups.

SUMMARY The shrimp Rimicaris exoculata swarms around hydrothermal black smoker chimneys at most vent sites along the Mid-Atlantic Ridge. This species maintains close proximity to the hydrothermal fluid, where temperatures can reach 350°C and steep thermal and chemical gradients are expected. We performed in vivo experiments in pressurized aquaria to determine the upper thermal limit [critical thermal maximum ( CT max )] of R. exoculata and to investigate some characteristics of the shrimp stress response to heat exposure. These experiments showed that the shrimp does not tolerate sustained exposure to temperatures in the 33-37°C range( CT max ). A heat-inducible stress protein belonging to the hsp70 family was identified in R. exoculata , and its synthesis threshold induction temperature is below 25°C. The R. exoculata optimal thermal habitat may thus be restricted to values lower than previously expected (<25°C).

SUMMARY We assessed the activation of p38-MAPK (mitogen-activated protein kinase) by osmotic and thermal stresses in the isolated perfused amphibian ( Rana ridibunda ) heart. Hyperosmotic stress induced the rapid activation of the kinase. In particular, in the presence of 0.5 mol l –1 sorbitol, p38-MAPK was maximally phosphorylated (by approximately twelvefold) at 15 min, while excess of NaCl (206 mmol l –1 final concentration) or KCl (16 mmol l –1 final concentration) stimulated a less potent activation, maximised (by approximately eightfold and fourfold) within 2 min and 30 s, respectively, relative to control values. The effect of all three compounds examined was reversible, since the kinase phosphorylation levels decreased upon reperfusion of the heart with normal bicarbonate-buffered saline. Conversely, hypotonicity did not induce any p38-MAPK activation. Furthermore, both hypothermia and hyperthermia induced considerable phosphorylation of the kinase, by four- and 7.5-fold, respectively, relative to control values. Immunohistochemical studies elucidated the localisation pattern of phospho-p38-MAPK and also revealed enhanced atrial natriuretic peptide (ANP) immunoreactivity in osmotically stressed hearts. Interestingly, SB 203580 (1 μmol l –1 ) not only completely blocked the activation of p38-MAPK by all these interventions, but also abolished the enhanced ANP immunoreactivity induced by 0.5 mol l –1 sorbitol. These findings indicate the possible involvement of ANP in the mechanisms regulating responses under such stressful conditions.