nystatin-a1 and Hypoxia

Topics: Adaptation, Physiological; Age Factors; Animals; Animals, Newborn; Digoxin; Energy Metabolism; Furosemide; Growth; Humans; Hypoxia; Infant, Newborn; Nystatin; Ouabain; Oxygen Consumption; Potassium Channels; Rats; Seals, Earless; Sheep; Sodium-Potassium-Exchanging ATPase; Swine; Turtles

Cytosolic free Ca (Caf) was measured in three different preparations of freshly prepared proximal tubules from the rabbit kidney during energy deprivation using fura-2. Isolated perfused tubules, tubules immobilized on glass cover slips, and tubules in suspension were subjected to inhibitors of oxidative phosphorylation ("chemical hypoxia"); the latter two preparations were also subjected to 40 min of anoxia. During normoxia, Caf ranged from 100 to 180 nM in all three preparations, and chemical hypoxia caused either no change or a small (30-100%) increase in Caf values. Subsequent addition of Ca ionophores increased Caf to 300-500 nM in the first 2 min and to greater than 1 microM after 15 min. In individual experiments, anoxia produced similar responses to those of chemical hypoxia, eliciting no average significant change in Caf, despite clear evidence for impaired respiration and plasma membrane damage after 40 min of anoxia. This lack of change in Caf was unrelated to "Ca buffering" by fura-2 or inactivation of the dye, since Caf increased to 666 +/- 59 nM upon addition of Ca ionophore during anoxia. These data suggest that increased Caf is not a prerequisite for cellular damage during anoxia in proximal renal tubules. Furthermore, no apparent alteration in plasma membrane permeability to Ca occurs before membrane disruption. Decreased ATP seems to initiate a series of Caf-independent events that cause irreversible injury.

Topics: Animals; Calcium; Cytosol; Female; Fluorescent Dyes; Fura-2; Hypoxia; In Vitro Techniques; Ionomycin; Kidney Tubules; Nystatin; Ouabain; Oxygen; Oxygen Consumption; Perfusion; Rabbits; Rotenone; Spectrometry, Fluorescence

When a suspension of rabbit proximal tubules is subjected to anoxia, ATP falls by 80-90% during 40 min of anoxia, and upon reoxygenation (reox) the cells only recover 25-50% of their initial ATP. Addition of Mg-ATP (magnesium chloride-treated ATP), Mg-ADP, or Mg-AMP (five aliquots of 200 nmol/ml added 10 min apart) during anoxia causes complete recovery of ATP levels, and respiratory and transport function after 40 min of reox. Similar additions of adenosine (ADO), or inosine (INO), or Mg-ATP only during reox are less effective. Lactate dehydrogenase (LDH) release after 40 min of anoxia is 30-40% under control conditions, only 10-15% when adenine nucleotides or ADO are added during anoxia, and 20% when INO is added, suggesting that these additions may stabilize the plasma membrane during anoxia and help preserve cellular integrity. During reox, recovery may depend on the entry of ATP precursors and, therefore, we explored the mechanism whereby exogenous ATP increases the intracellular ATP content. Additions of Mg-ATP, Mg-ADP, or Mg-AMP to continuously oxygenated tubules increase cellular ATP content three- to fourfold in 1 h. The added ATP and ADP are rapidly degraded to AMP, and more slowly to ADO, INO, and hypoxanthine. Furthermore, the ATP-induced increase in cellular ATP is abolished by the exogenous addition of adenosine deaminase, which converts extracellular ADO to INO. These results suggest that the increase in cellular ATP requires extracellular ADO. The ADO obtained from the breakdown of AMP may be preferentially transported into the renal cells to be resynthesized into cellular AMP and ATP.

Topics: Adenine Nucleotides; Adenosine Deaminase; Animals; Extracellular Space; Hypoxanthine; Hypoxanthines; Hypoxia; Ischemia; Kidney; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Nystatin; Oxygen Consumption; Potassium; Rabbits; Time Factors

The effects of short-term anoxia and hypoxia were studied in a rabbit proximal renal tubule suspension in order to avoid the hemodynamic consequences of clamp-induced ischemia. The suspension was subjected to anoxia for 10-40 min and the effects on a number of cellular transport and respiratory parameters were monitored. Cellular respiration was measured upon addition of nystatin (Nys) to maximally stimulate Na pump activity. Mitochondrial respiration was measured in the tubules by addition of digitonin and ADP to obtain the state 3 respiratory rate. The release of lactate dehydrogenase (LDH) was measured as an index of plasma membrane damage. The cellular contents of K and Ca were also measured. Results show that 10 and 20 min of anoxia partially inhibited Nys-stimulated and mitochondrial respiration, and partially decreased the K contents, but all these effects were largely reversible after 20 min of reoxygenation. After 40 min of anoxia and 20 min of reoxygenation, all these variables remained irreversibly inhibited: Nys-stimulated respiration by 54%, mitochondrial respiration by 50%, K content by 42%, and LDH release was 40% of total. Ca content decreased slightly during anoxia, but increased up to fourfold during severe hypoxia; the excess Ca was released during the first 10 min of reoxygenation. The degree of respiratory impairment was identical during anoxia or hypoxia, suggesting that Ca accumulation was not associated with the impairment. Decreasing the extracellular Ca to 2.5 microM decreased LDH release significantly during anoxia, suggesting that plasma membrane damage during anoxia may be associated with increased intracellular free Ca. Addition of Mg-adenosine triphosphate during anoxia dramatically improved recovery of all the measured parameters after the anoxic period.

Topics: Adenosine Triphosphate; Animals; Calcium; Hypoxia; Ischemia; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Nystatin; Oxygen Consumption; Potassium; Rabbits