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Frederich, Markus, and Hans O. Pö rtner. Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado. Am J Physiol Regulatory Integrative Comp Physiol 279: R1531-R1538, 2000.-Geographic distribution limits of ectothermal animals appear to be correlated with thermal tolerance thresholds previously identified from the onset of anaerobic metabolism. Transition to these critical temperatures was investigated in the spider crab (Maja squinado) with the goal of identifying the physiological processes limiting thermal tolerance. Heart and ventilation rates as well as PO 2 in the hemolymph were recorded on-line during progressive temperature change between 12 and 0°C (1°C/h) and between 12 and 40°C (2°C/h). Lactate and succinate were measured in tissues and hemolymph after intermediate or final temperatures were reached. High levels of hemolymph oxygenation suggest that an optimum range of aerobic performance exists between 8 and 17°C. Thermal limitation may already set in at the transition from optimum to pejus (pejus ϭ turning worse, progressively deleterious) range, characterized by the onset of a decrease in arterial PO 2 due to reduced ventilatory and cardiac performance. Hemolymph PO 2 values fell progressively toward both low and high temperature extremes until critical temperatures were reached at ϳ1 and 30°C, as indicated by low PO 2 and the onset of anaerobic energy production by mitochondria. In conclusion, the limited capacity of ventilation and circulation at extreme temperatures causes insufficient O 2 supply, thereby limiting aerobic scope and, finally, thermal tolerance. aerobic capacity; anaerobic metabolism; optodes; partial pressure of oxygen CRITICAL TEMPERATURES (T c ) have been defined for different marine invertebrate and fish species as being characterized by the onset of anaerobic metabolism, which is caused by a mismatch of O 2 demand and O 2 supply (for review, see Refs. 35 and 36). Extended exposure to temperatures above high T c or below low T c finally leads to death of the animal unless thermal acclimation, i.e., a shift of T c values, occurs (40, 51). One hypothesis is that the adjustment of mitochondrial density and capacity is involved in setting thermal tolerance limits and is therefore related to geographic distribution (35, 36). As a consequence, the relationship between O 2 availability to tissues and O 2 demand appears to be crucial for survival of exposure to temperature extremes. Study of the processes of O 2 uptake by ventilation and O 2 distribution by circulation therefore appear important to further our understanding of the O 2 limitation of thermal tolerance. Therefore, we chose to study these systemic aspects of thermal tolerance, selecting a crustacean, Maja squinado (Herbst), as a model organism. As yet, physiological studies of crustaceans have addressed temperature effects on O 2 consumption, heart and ventilatory performance, or growth (12-14, 16, 23, 34, 44). In most of these studies, temperatures were chosen wit...

SUMMARYExposure of marine invertebrates to high temperatures leads to a switch from aerobic to anaerobic metabolism, a drop in the cellular ATP concentration ([ATP]), and subsequent death. In mammals, AMP-activated protein kinase (AMPK) is a major regulator of cellular [ATP] and activates ATP-producing pathways, while inhibiting ATP-consuming pathways. We hypothesized that temperature stress in marine invertebrates activates AMPK to provide adequate concentrations of ATP at increased but sublethal temperatures and that AMPK consequently can serve as a stress indicator (similar to heat shock proteins, HSPs). We tested these hypotheses through two experiments with the rock crab, Cancer irroratus. First, crabs were exposed to a progressive temperature increase (6°C h -1 ) from 12 to 30°C. AMPK activity, total AMPK protein and HSP70 levels, reaction time, heart rate and lactate accumulation were measured in hearts at 2°C increments. AMPK activity remained constant between 12 and 18°C, but increased up to 9.1(±1.5)-fold between 18 and 30°C. The crabsʼ reaction time also decreased above 18°C. By contrast, HSP70 (total and inducible) and total AMPK protein expression levels did not vary significantly over this temperature range. Second, crabs were exposed for up to 6 h to the sublethal temperature of 26°C. This prolonged exposure led to a constant elevation of AMPK activity and levels of HSP70 mRNA. AMPK mRNA continuously increased, indicating an additional response in gene expression. We conclude that AMPK is an earlier indicator of temperature stress in rock crabs than HSP70, especially during the initial response to high temperatures. We discuss the temperature-dependent increase in AMPK activity in the context of Shelfordʼs law of tolerance. Specifically, we describe AMPK activity as a cellular marker that indicates a thermal threshold, called the pejus temperature, T p . At T p the animals leave their optimum range and enter a temperature range with a limited aerobic scope for exercise. This T p is reached periodically during annual temperature fluctuations and has higher biological significance than earlier described critical temperatures, at which the animals switch to anaerobic metabolism and HSP expression is induced.

AMP-activated protein kinase (AMPK)1 and AMPK kinase comprise a protein kinase cascade that has been highly conserved throughout evolution (1, 2). Increases in AMP concentration ([AMP]) activate this cascade by four mechanisms (3-5). These mechanisms are as follows: 1) an allosteric activation by AMP of AMPK kinase, which then phosphorylates AMPK; 2) the binding of AMP to AMPK, which makes it a poorer substrate for protein phosphatases; 3) the binding of AMP to AMPK, which makes AMPK a better substrate for AMPK kinase; and 4) the allosteric activation by AMP of AMPK. The activating effects of AMP are antagonized by high concentrations of ATP. Since the AMPK is activated when AMP is elevated and ATP is depressed, AMPK is hypothesized to act as cellular "fuel gauge" (1).After AMPK activation, in response to metabolic stress, AMPK phosphorylates enzymes, leading to activation of catabolic pathways to increase ATP synthesis and inhibition of anabolic pathways to limit ATP consumption. For example, AMPK phosphorylation decreases acetyl-CoA-carboxylase (ACC) activity, which decreases malonyl-CoA concentration. Malonyl-CoA inhibits carnitine palmitoyltransferase-1, which transports fatty acids into the mitochondrion (6, 7). Reduction of malonyl-CoA increases fatty acid uptake via carnitine palmitoyltransferase-1 and, thereby, the fatty acid oxidation by the mitochondria, which increases ATP production. In skeletal muscle and hearts, AMPK activity also increases glucose uptake by enhancing GLUT-4 translocation (8 -10). In hearts, AMPK phosphorylation of 6-phosphofructo-2-kinase increases fructose 2,6-bisphosphate, a stimulator of 6-phosphofructo-1-kinase, which accelerates glycolysis (11). AMPK also downregulates ATP-consuming pathways, such as glycogen, cholesterol, and fatty acid synthesis (12, 13). The aim of the present study was to define the relationship between AMPK activation and cytosolic [AMP] in vivo in the isolated perfused rat heart. To accomplish this, we employed an approach that restrains substrate metabolism by use of the inhibitors bromo-octanoate (BrO) to inhibit fatty acid oxidation (16) and amino-oxyacetate (AOA), to inhibit pyruvate oxidation (17). Using this approach together with increased work demand reduced the phosphocreatine (PCr) (18). We used 31 P NMR spectroscopy to measure the PCr and ATP content as well as the intracellular pH (pH i ) of these hearts. The creatine kinase and the adenylate kinase equilibrium expressions were used to calculate [ADP] and [AMP], respectively. The net effect of [PCr] reductions is an increase in [AMP] with a relatively constant [ATP]. Cytosolic [AMP] was therefore manipulated in a relatively controlled manner, and the AMPK activity was measured. EXPERIMENTAL PROCEDURESPreparation of Isolated Perfused Rat Hearts-Hearts of male Sprague-Dawley rats (280 -320 g) were isolated and perfused in the isovolumic Langendorf model (18). The Krebs-Henseleit buffer (KH) perfusate contained 118 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 1.75 mM CaCl 2 , 0.5 mM ...