Metabolic depression is delayed and mitochondrial impairment averted during prolonged anoxia in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935)

Lepidophthalmus louisianensis burrows deeply into oxygen-limited estuarine sediments and is subjected to extended anoxia at low tides. Large specimens (> 2 g) have a lethal time for 50% mortality (LT₅₀) of 64 h under anoxia at 25 °C. Small specimens (< 1 g) have a significantly higher LT₅₀ of 113 h, which is the longest ever reported for a crustacean. Whole body lactate levels rise dramatically under anoxia and exceed 120 μmol g.f.w.^− 1 by 72 h. ATP, ADP, and AMP do not change during 48 h of anoxia, but arginine phosphate declines by over 50%. Thus arginine phosphate may help stabilize the ATP pool. Surprisingly, when compared to the aerobic resting rate, ATP production under anoxia declines only moderately during the first 12 h, and drops to only about 30% between 12 and 48 h. Finally, after 48 h of anoxia, a major metabolic depression to less than 5% occurs. Downregulation of metabolism is delayed in L. louisianensis compared to many invertebrates that exhibit facultative anaerobiosis. Bioenergetic constraints as a result of eventual metabolic depression lead to ionic disturbances like calcium overload and compromised membrane potential of mitochondria. Because these phenomena trigger apoptosis in mammalian species, we evaluated the susceptibility of ghost shrimp mitochondria to opening of the mitochondrial permeability transition pore (MPTP) and associated damage. Energized mitochondria isolated from hepatopancreas possess a pronounced capacity for calcium uptake. Exogenous calcium does not stimulate opening of the MPTP, which potentially could reduce cell death during prolonged anoxia.     

Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog,Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (K_m = 0.098 ± 0.003 and K_m = 0.092 ± 0.008) than PK from control and anoxic conditions (K_m = 0.065 ± 0.003 and K_m = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.     

Protein kinase and phosphatase responses to anoxia in crayfish, Orconectes virilis: purification and characterization of cAMP-dependent protein kinase

The freshwater crayfish, Orconectes virilis, shows good anoxia tolerance, enduring 20 h in N₂-bubbled water at 15°C. Metabolic responses to anoxia by tolerant species often include reversible phosphorylation control over selected enzymes. To analyze the role of serine/threonine kinases and phosphatases in signal transduction during anoxia in O. virilis, changes in the activities of cAMP-dependent protein kinase (PKA) and protein phosphatases 1, 2A, and 2C were measured in tail muscle and hepatopancreas over a time course of exposure to N₂-bubbled water. A strong increase in the percentage of PKA present as the free catalytic subunit (% PKAc) occurred between 1 and 2 h of anoxia exposure whereas phosphatase activities were strongly reduced. This suggests that PKA-mediated events are important in the initial response by tissues to declining oxygen availability. As oxygen deprivation became severe and prolonged (5–20 h) these changes reversed; the % PKAc fell to below control values and activities of phosphatases returned to or rose above control values. Subcellular fractionation also showed a decrease in PKA associated with the plasma membrane after 20 h anoxia whereas cytosolic PKA content increased. PKAc purified from tail muscle showed a molecular weight of 43.8±0.4 kDa, a pH optimum of 6.8, a high affinity for Mg ATP (K_m=131.0±14.4 μM) and Kemptide (K_m=31.6±5.2 μM). Crayfish PKAc was sensitive to temperature change; a break in the Arrhenius plot occurred at approximately 15°C with a 2.5-fold rise in activation energy at temperatures <15°C. These studies demonstrate a role for serine/threonine protein kinases and phosphatases in the metabolic adjustments to oxygen depletion by crayfish organs.     

Effects of anoxia and post-anoxia recovery on carbohydrate metabolism in the jaw muscle of the crab Chasmagnathus granulatus maintained on carbohydrate-rich or high-protein diets

The present study assesses the effects of 1-h anoxia and 3-h post-anoxia recovery period on the activity of pyruvate kinase (PK), ¹⁴CO₂ produced from ¹⁴C-glucose and ¹⁴C-lactate, ATP, and glycogen levels in the jaw muscle of Chasmagnathus granulatus fed either a carbohydrate-rich (HC) or high-protein (HP) diet.In the HC control group the jaw muscle PK activity was higher than in HP-fed crabs. In jaw muscle from control HP-fed crabs the lactate oxidation was higher than in HC-fed animals. We observed increased PK activity and ATP concentration and a reduction in the glycogen concentration, ¹⁴CO₂ production from ¹⁴C-lactate in HP-fed crab jaw muscle during anoxia. In crabs fed an HC diet the PK activity decreased in anoxia, the ¹⁴CO₂ production from ¹⁴C-glucose increased, and the ¹⁴CO₂ production from ¹⁴C-lactate did not change.During recovery, a low oxidation capacity for lactate was found in jaw muscle of both dietary groups. PK activity and ¹⁴CO₂ production from ¹⁴C-glucose were high during the recovery period only in the jaw muscle from HP-fed crabs.Recovery decreased the ATP concentration of both dietary groups as compared to anoxia and normoxia, and did not restore the glycogen concentration in the jaw muscle.     

Activation of nuclear protein binding to the antioxidant/electrophile response element in vascular smooth muscle cells by benzo(a)pyrene

This laboratory has previously shown that binding of nuclear proteins to the antioxidant/electrophile response element (ARE/EpRE) participates in deregulation of vascular gene expression by benzo(a)pyrene (BaP), a suspected atherogen. In the present study, oligonucleotides representing ARE/EpREs within the c-Ha-ras and glutathione-S-transferase (GST-Ya) promoters were employed to evaluate the role of flanking sequences in stabilizing protein:DNA interactions in BaP-treated vascular smooth muscle cells (vSMCs). We also wanted to define promoter-specific patterns of protein recognition to ARE/EpREs in this cell system. In electrophoretic mobility shift assays (EMSA), optimal protein binding to a human Ha-ras ARE/EpRE variant sequence fitted to match the extended mouse(m) GST-Ya ARE/EpRE core (5'-TMAnnRTGAYnnnGCR-3') was dependent on 5' nucleic acid sequence. Using immobilized DNA affinity chromatography (IDAC), we identified four nuclear proteins of M(r) 62, 60, 50, and 30 kDa that associated specifically with the mGSTYa ARE/EpRE. Photo crosslinking to a BrdU-substituted hHa-ras or mGST ARE/EpRE probe identified specific proteins of M(r) 80, 60, 55, 25, 23 kDa or 80, 60, 55, 27, 25, 23 kDa, respectively. Protein:DNA complexes detected using IDAC eluate overlapped with those observed in crude nuclear extracts. Chemical treatments known to modulate ARE/EpRE protein binding in vSMCs did not alter overall protein:DNA affinity and/or sequence recognition to either hHa-ras or mGST-Ya elements. We conclude that nucleotide sequences 5' to the core ARE/EpRE influence specific binding of nuclear proteins and that multiple proteins bind to ARE/EpREs in a promoter-specific manner in vSMCs.