Reversible decreases in ATP and PCr concentrations in anoxic turtle brain

A hallmark of anoxia tolerance in western painted turtles is relative constancy of tissue adenylate concentrations during periods of oxygen limitation. During anoxia heart and brain intracellular compartments become more acidic and cellular energy demands are met by anaerobic glycolysis. Because changes in adenylates and pH during anoxic stress could represent important signals triggering metabolic and ion channel down-regulation we measured PCr, ATP and intracellular pH in turtle brain sheets throughout a 3-h anoxic-re-oxygenation transition with ³¹P NMR. Within 30 min of anoxia, PCr levels decrease 40% and remain at this level during anoxia. A different profile is observed for ATP, with a statistically significant decrease of 23% occurring gradually during 110 min of anoxic perfusion. Intracellular pH decreases significantly with the onset of anoxia, from 7.2 to 6.6 within 50 min. Upon re-oxygenation PCr, ATP and intracellular pH recover to pre-anoxic levels within 60 min. This is the first demonstration of a sustained reversible decrease in ATP levels with anoxia in turtle brain. The observed changes in pH and adenylates, and a probable concomitant increase in adenosine, may represent important metabolic signals during anoxia.     

Effects of Fructose-1,6-Bisphosphate on Glutamate Release and ATP Loss from Rat Brain Slices During Hypoxia

Abstract: Fructose-1,6-bisphosphate (FBP), an intermediate of glucose metabolism, is neuroprotective in brain hypoxia or ischemia. Because the mechanisms for this protection are not clear, we examined the effects of FBP on two important events in brain ischemia, i.e., loss of ATP and release of the excitatory neurotransmitter glutamate. Glutamate release from cortical brain slices was measured fluorometrically (glutamate dehydrogenase)-catalyzed conversion of glutamate to α-ketoglutarate) during hypoxia (Po₂ 15 mm Hg) or hypoxia plus 100 µ M cyanide. FBP (3.5 m M , with glucose 20 m M ) reduced glutamate release during hypoxia by 55% and during hypoxia/cyanide by 46% ( p < 0.005), and prevented a significant fall in [ATP]. [ATP] was maintained in oxygenated glucose-free conditions with 20 but not 3.5 m M FBP, and fell to <20% of normal with hypoxia. Despite the drop in [ATP], 3.5 or 20 m M FBP without glucose decreased hypoxia-evoked glutamate release. We conclude (1) FBP present without glucose preserves normal [ATP] only when oxygen is available, suggesting limited uptake and metabolism; and (2) FBP decreases hypoxia-evoked glutamate release by processes independent of [ATP]. These results suggest protective actions of FBP that are separate from augmentation of anaerobic energy production, as previously proposed.     

Isolation and characterization of human urine extracellular vesicles

Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO₄ prior to vesicle isolation by differential centrifugation. Treatment with ZnSO₄ did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers.     

Kidney and bladder function in a uricotelic treefrog (Phyllomedusa sauvagei)

Summary Phyllomedusa sauvagei, a xeric adapted treefrog, excretes large amounts of nitrogen as urate when fed insects, even when deprived of additional water. Most terrestrial anurans produce urea which they do not excrete when they are deprived of water. We investigated the differences in renal function underlying the unusual excretory capacities ofP. sauvagei. Glomerular filtration rates (GFR) were measured inP. sauvagei in water and when deprived of water, except that in food, for up to 27 days. For comparison a toad (Bufo boreas) was studied in water and during water deprivation. In water both species produced 30–40 ml urine kg^–1 h^–1 and resorbed only ca. 50% of the filtrate. With water deprivation, GFR rapidly approached zero inB. boreas, but remained high (20–40 ml kg^–1 h^–1) inP. sauvagei despite reductions in urine production of up to 100-fold. During water deprivation inP. sauvagei, urate excretion was between 250–300 moles kg^–1 h^–1 and 90% of this reflects net tubular secretion. Urate clearances were similar to those of para-amino hippurate, indicating effective removal of urate from the peritubular circulation. Urea, sodium and chloride showed net fractional resorptions of 98–99%, and 85% of the potassium was resorbed. At low rates of urine production, urine to plasma (U/P) ratios for inulin in bladder urine were 20–100 whereas those for ureteral urine were ca. 10. The urinary bladder also functions as a water reserve during dehydration.     

Hypoxia increases calcium flux through cortical neuron glutamate receptors via protein kinase C

The effects of 30 s to 10 min hypoxia (PO2-10 mmHg) on glutamate receptor activity were studied in murine cortical neurons. Receptor activity was assessed as a rise in intracellular calcium concentration ([Ca2+]i) following a 10 s application of 1 mm glutamate or 100 micro mN-methy-d-aspartate (NMDA) in the presence of 0.1 mm Mg2+ and 10 micro m glycine. Change in [Ca2+]i elicited by glutamate increased 26% (n = 192, p < 0.001) and that to NMDA by 74% (n = 9, p < 0.01) during a 100-s period of hypoxia. After 10 min hypoxia, responses to glutamate were 62% smaller than those in normoxia, with increased basal intracellular [Ca2+]i predicting reduced receptor activity. When neurons were exposed to NMDA after 10 min of hypoxia, [Ca2+]i increases were 12% smaller than after 100 s hypoxia, but still 53% larger than in oxygenated neurons (n = 9, p = 0.01). Neurons expressed relatively similar amounts of NR2A, -B, -C, and -D subunits. The phosphorylation of NMDA NR1 subunits increased during hypoxia. Pre-treatment of neurons with a protein kinase C (PKC) inhibitor (chelerythrine, 10 micro m) prevented increases in N-methy-d-aspartate receptor (NMDAR) activity during hypoxia and reduced the phosphorylation of NR1 subunits. These results suggest that enhancement of glutamate receptor activity during the first minutes of hypoxia is mediated by phosphorylation of NMDARs by PKC and that other mechanisms, possibly involving intracellular calcium, limit glutamate receptor-mediated calcium influx during longer periods of hypoxia.     

Period of irreversible therapeutic intervention during sepsis correlates with phase of innate immune dysfunction

Sepsis is a major health problem in the United States with high incidence and elevated patient care cost. Using an animal model of sepsis, cecum ligation, and puncture, we observed that mice became rapidly hypothermic reaching a threshold temperature of 28 °C within 5-10 h after initiation of the insult, resulting in a reliable predictor of mortality, which occurred within 30-72 h of the initial procedure. We also observed that the inflammatory gene expression in lung and liver developed early within 1-2 h of the insult, reaching maximum levels at 6 h, followed by a decline, approaching basal conditions within 20 h. This decrease in inflammatory gene expression at 20 h after cecal ligation and puncture was not due to resolution of the insult but rather was an immune dysfunction stage that was demonstrated by the inability of the animal to respond to a secondary external inflammatory stimulus. Removal of the injury source, ligated cecum, within 6 h of the initial insult resulted in increased survival, but not after 20 h of cecal ligation and puncture. We concluded that the therapeutic window for resolving sepsis is early after the initial insult and coincides with a stage of hyperinflammation that is followed by a condition of innate immune dysfunction in which reversion of the outcome is no longer possible.