The enzymatic measurement of adenine nucleotides and P-creatine in picomole amounts

Lust, W. David; Feussner, Gretchen K.; Barbehenn, Elizabeth; Passonneau, Janet V.

doi:10.1016/0003-2697(81)90144-5

Cited by 160 publications

(40 citation statements)

References 12 publications

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“…The cells and media were collected, rapidly frozen, and stored at Ϫ80°C. ATP intracellular concentrations were analyzed using a luciferase bioluminescence procedure previously described (33). Briefly, 10 l of cell extract were mixed with 50 l of ATP reagent [solution mixture: 0.5 ml of imidazole-HCl buffer (1 M), 0.02 ml of MgCl2 (1 M), and 0.750 ml of KCl (1 M)] and vortexed.…”

Section: Methodsmentioning

confidence: 99%

Role of nitric oxide in the regulation of HIF-1α expression during hypoxia

Agani

Puchowicz

Chávez

et al. 2002

American Journal of Physiology-Cell Physiology

128

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Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of HIF-1α and HIF-1β subunits, controls the expression of a large number of genes involved in the regulation of cellular responses to reduced oxygen availability. The oxygen-regulated subunit, HIF-1α, is stabilized in cells exposed to hypoxia. The regulation of hypoxic responses by nitric oxide (NO) is believed to have wide pathophysiological relevance, thus we investigated whether NO affects HIF-1 activation in hypoxic cells. Here we show that NO generated from NO donors prevented HIF-1α hypoxic accumulation in Hep 3B and PC-12 cells. Addition of a glutathione analog or peroxynitrite scavengers prevented the NO-induced inhibition of HIF-1α accumulation in both cell lines. Exposure to NO was associated with inhibition of mitochondrial electron transport and compensatory glycolysis, which maintained normal cellular ATP content. Succinate, a Krebs cycle intermediate and respiratory chain substrate, restored HIF-1α hypoxic induction in the cells, suggesting involvement of mitochondria in regulation of HIF-1α accumulation during hypoxia. Regulation of HIF-1α by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia in mammalian cells.

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Section: Methodsmentioning

confidence: 99%

Role of nitric oxide in the regulation of HIF-1α expression during hypoxia

Agani

Puchowicz

Chávez

et al. 2002

American Journal of Physiology-Cell Physiology

128

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“…After anoxia or an equivalent time period the slices were frozen rapidly, lyophilized and the CAI regions dissected and weighed as described above. The ATP was then extracted by homogenizing the tissue in 3 N ice-cold perchloric acid and measured, after neutralization, using the firefly luciferin-luciferase assay (Lust, Feussner, Barbehenn & Passonneau, 1982). Sodium and potassium levels were measured in the dissected CAI region after 5 min of anoxia.…”

Section: Methodsmentioning

confidence: 99%

The importance of sodium for anoxic transmission damage in rat hippocampal slices: mechanisms of protection by lidocaine.

Fried

Amorim

Chambers

et al. 1995

The Journal of Physiology

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1. The effect of sodium influx on anoxic damage was investigated in rat hippocampal slices.Previous experiments demonstrated that a concentration of tetrodotoxin which blocks neuronal transmission protects against anoxic damage. In this study we examined low concentrations of lidocaine (lignocaine; which do not block neuronal transmission), for their effect on recovery of the evoked population spike recorded from the CAl pyramidal cell layer. 2. Recovery of the population spike, measured 60 min after a 5 min anoxic period, was 4 + 2% of its preanoxic, predrug level. Lidocaine concentrations of 10, 50, and 100 /M significantly improved recovery to 56 + 12, 80 + 7 and 70 + 14 %, respectively. 3. Lidocaine (10 /M) did not alter the size of the evoked response before anoxia and had no significant effect on potassium levels or calcium influx during anoxia. It did, however, reduce cellular sodium levels (146 + 7 vs. 202 + 12 nmol mg-) and preserve ATP levels (2-17 + 0 07 vs. 1-78 + 0 07 nmol mg-1) during anoxia. All values were measured at the end of 5 min of anoxia except those for Ca2+ influx which were measured during 10 min of anoxia. 4. High concentrations of lidocaine (100 /M) did not improve recovery significantly over that observed with 10 jtM. They also had no significantly greater effects on sodium levels than 10/SM lidocaine (137 ± 12 vs. 146 + 7 nmol mg-); however, 100 /M lidocaine significantly improved potassium (202 + 18 vs. 145 + 6 nmol mg-1) and ATP (2-57 + 0-06 vs. 2-17 + 0 07 nmol mg-') levels, while reducing calcium influx (7-76 + 0-12 vs. 9X24 + 0 39 nmol mg-1 (10 min)-1) when compared with 10 /M lidocaine. 5. We conclude that sodium influx and ATP depletion are of major importance in anoxic damage since 10 uM lidocaine reduced these changes during anoxia and improved recovery of the population spike. In addition, our results indicate that the properties of the sodium channel are altered during anoxia, since sodium influx is blocked by a concentration of lidocaine that does not affect the population spike in the preanoxic period.Reduced oxygen delivery to the brain can lead to permanent loss of brain function (Hansen, 1985;Siesjo, 1988). It is important to understand the mechanism of this damage if one is to protect neurons. Studies have implicated calcium

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“…The latter included the region from the stratum radiatum to the alveus including the CAI pyramidal cell layer. High-energy nucleotides were measured using the firefly luciferin-luciferase assay (Lust, Feussner, Barbehenn & Passonneau, 1982).…”

Section: Methodsmentioning

confidence: 99%

Calcium and long‐term transmission damage following anoxia in dentate gyrus and CA1 regions of the rat hippocampal slice.

Kass¹,

Lipton²

1986

The Journal of Physiology

141

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SUMMARY1. The mechanism oflong-term anoxic damage in brain tissue is investigated using the rat hippocampal slice as a model system. The effects of short durations of anoxia on subsequent transmission through two neural pathways are studied. 10 min of anoxia irreversibly abolishes transmission between the perforant path and the dentate granule cells while only 7 min of anoxia irreversibly abolishes transmission between the Schaeffer collaterals and the CAI pyramidal cells. We examine the involvement ofCa2+ in this irreversible transmission damage and, also, the differential sensitivities of the dentate gyrus and CAI regions.2. Substitution of a buffer containing 0 Ca2+ and 10 mM-Mg2+ during the anoxic period substantially improves the recovery of synaptic transmission in both regions of the slice. Dentate gyrus transmission recovers completely after 20 min of anoxia and CAI transmission survives 10 min of anoxia. These results suggest that Ca2+ influx during anoxia may be an important cause of the long-term damage.3. The uptake of 45Ca2+ into the intracellular space of the slice is increased during anoxia. This effect is approximately twice as large in CAI as in the dentate gyrus. Thus, in the dentate gyrus the calculated exchangeable pool of Ca2+ is increased 30% by anoxia and in the CAI it is increased by 70 %. 4. Two incubating conditions which decrease the amount of 45Ca2+ uptake during anoxia protect transmission against long-term damage. (a) Pre-incubation of the slices with 25 mM-creatine elevates tissue phosphocreatine and attenuates the fall in adenosine 5'-triphosphate (ATP) during anoxia. This is associated with partial protection against transmission damage and an approximate 50 % attenuation of the anoxic uptake of 45Ca2+. (b) Inclusion of 2 mM-cobalt in the buffer reduces the normoxic uptake of 45Ca2+ so that the uptake during anoxia is no greater than normoxic uptake in the absence of cobalt. This is associated with a complete protection against long-term transmission damage following 10 min of anoxia in the dentate gyrus.5. A kinetic analysis of the 45Ca2+ uptake shows that the anoxic uptake results primarily from inhibition of the unidirectional efflux of Ca2+ from the cells; there is no calculable increase in the undirectional influx. This suggests that anoxia increases I. S. KASS AND P. LIPTON Ca2+ uptake by inhibiting one or more Ca2+-extrusion processes and not by opening depolarization-sensitive Ca2+ channels. This inference is supported by the action of the 2 mM-cobalt. While this ion almost completely blocks veratrine-induced 45Ca2+ uptake it has no effect on the anoxic increase in 4-Ca2+ uptake in the dentate gyrus and only a 15 % inhibitory effect on the anoxic increase in 45Ca2+ uptake into CAl.6. 3 x 10-6 M-ouabain decreases cell K+ and elevates cell Na+ more than does 10 min of anoxia yet it has no effect on 45Ca2+ uptake. This (a) supports the inference that depolarization during anoxia is not the factor leading to increased Ca2+ uptake and (b) suggests that the Na+-Ca2+ exchange me...

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The enzymatic measurement of adenine nucleotides and P-creatine in picomole amounts

Cited by 160 publications

References 12 publications

Role of nitric oxide in the regulation of HIF-1α expression during hypoxia

Role of nitric oxide in the regulation of HIF-1α expression during hypoxia

The importance of sodium for anoxic transmission damage in rat hippocampal slices: mechanisms of protection by lidocaine.

Calcium and long‐term transmission damage following anoxia in dentate gyrus and CA1 regions of the rat hippocampal slice.

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