Ultrastructure of tubules subjected to hypoxia/reoxygenation without and with αKG/ASP. Tubules were subjected to 60 minutes of hypoxic incubation and 60 minutes of reoxygenation without (a) or with supplemental αKG/ASP only during reoxygenation (b). ATP was added to the medium during reoxygenation. Each picture is representative of observations that have been made on multiple tubule preparations. Original magnification, ×7000.

Immunoblot for total β1 integrin. Tubules were incubated under oxygenated control conditions for 75 minutes (75′ TC), for 60 minutes of hypoxia, or 60 minutes of hypoxia and 60 minutes reoxygenation with NES during either during hypoxia or reoxygenation. H, sample taken at the end of 60 minutes hypoxia; R, sample taken after 60 minutes hypoxia followed by 60 minutes reoxygenation. ATP was added to the medium during reoxygenation.

Immunolocalization of pY and paxillin. Tubules were from the same experiments as illustrated in Figure 3 ▶ and the images are similarly organized. In each set, a and d are oxygenated time controls. b and c: Representative of the range of changes seen in tubules at the end of 60 minutes of hypoxia with NES. e: Representative tubules after hypoxia followed by reoxygenation with NES. f: Paired flasks that were also subjected to hypoxia and reoxygenation, but were supplemented with αKG/ASP during reoxygenation. Magnifications are the same in each panel; scale bars,10 μm.

Effect of inhibiting tyrosine phosphatases with pervanadate on dephosphorylation during hypoxia. Tubules were subjected to 60 minutes of hypoxia (H) with NES without or with 120 μmol/L of pervanadate (PERV). Samples collected at the end of hypoxia were immunoblotted for pY (A) and paxillin (B) and immunostained for pY (C). TC indicates oxygenated time control samples corresponding to the start of hypoxia at 15 minutes of incubation and the end of hypoxia at 75 minutes of incubation. Each lane in the immunoblot is from a separate flask subjected to the indicated conditions.

Tyrosine phosphorylation patterns of focal adhesion proteins during hypoxia/reoxygenation. These samples correspond to some of the conditions studied in Figure 1 ▶ and were taken from the same experiments. A to E: Western blots probed with the indicated mAbs of whole cell extracts from tubules subjected to 60 minutes of hypoxia and reoxygenation with either NES or αKG/ASP. pY, Phosphotyrosine; FAK, focal adhesion kinase; H, samples taken at the end of 60 minutes hypoxia; R, sample taken from the same flask as the H sample in the preceding lane enclosed in the same box, but obtained after 60 minutes of reoxygenation. αKG/ASP (4.0 mmol/L) was present during either only hypoxia (lane 4) or only reoxygenation (lane 7) as indicated; TC, oxygenated time-control tubules sampled after the indicated durations of incubation.

Cytoskeletal alterations during hypoxia/reoxygenation. Tubules were subjected to 60 minutes of hypoxic incubation followed by 60 minutes of reoxygenation without or with supplemental αKG/ASP only during reoxygenation. ATP was added to the medium during reoxygenation. Samples fixed in paraformaldehyde-lysine-periodate at the end of hypoxia or after hypoxia and reoxygenation were cryosectioned and stained for F-actin with rhodamine phalloidin or immunostained for fodrin, or the β1 or α6 integrin subunits. In each of the sets of images, a and d are oxygenated time controls corresponding to the ends of the 60-minute hypoxic and 60-minute hypoxia plus 60 minutes reoxygenation periods, respectively. b and c are representative of the range of changes seen in tubules at the end of the 60-minute hypoxic period. The e panels show representative tubules after hypoxia followed by reoxygenation with NES. The f panels are from the paired flasks that were also subjected to hypoxia and reoxygenation, but were supplemented with αKG/ASP during reoxygenation. Magnifications are the same in each panel; scale bars, 10 μm.

Quantitation of pY immunostaining intensity patterns during hypoxia/reoxygenation. Tubules were immunostained for pY as in Figure 8 ▶ after 135 minutes of oxygenated control incubation (135′ TC), 60 minutes hypoxia with no extra substrates (H NES), 60 minutes of hypoxia and 60 minutes of reoxygenation with no extra substrates (R NES), or 60 minutes hypoxia and 60 minutes of reoxygenation with αKG/ASP during reoxygenation (R αKG/ASP). Shown are the percentages of tubules with entirely normal, good, weak, or absent staining among a total of 40 to 70 tubules from three separate preparations that were examined for each condition. By these criteria, a and f of the pY-stained tubules in Figure 8 ▶ show normal tubules. d: A good tubule, which is a common finding under control conditions and is characterized by staining that is weaker or more irregular than the best tubules seen, usually because of the angle of sectioning. b and e: Tubules with weak staining. Staining would be classified as absent for the tubule in c.

Prevention and reversal by αKG/ASP of the energetic deficit developing in tubules during hypoxia/reoxygenation. Tubules were subjected to 60 minutes hypoxia and 60 minutes reoxygenation with either no extra substrate (NES), or 4 mmol/L of α-ketoglutarate plus 4 mmol/L aspartate (αKG/ASP) during only reoxygenation (R), during only hypoxia (H), or during hypoxia and reoxygenation (H and R). Reoxygenation was studied both without and with supplemental exogenous purine (250 μmol/L ATP). Oxygenated control tubules were incubated for the same total durations as the corresponding hypoxia/reoxygenation groups. ATP was added to the medium of the purine-supplemented, oxygenated control group for the last 60 minutes of incubation. As in all studies, the incubation medium for all flasks contained glucose, lactate, alanine, and butyrate during both hypoxia and reoxygenation. Cell ATP levels sampled at the end of hypoxia and at the end of reoxygenation are means ± SE for n = 5. *, Significantly different from corresponding NES group; #, significantly different from corresponding “R only” group.

Quantitation of changes of tyrosine phosphorylation during hypoxia. A: Representative scan as used for densitometry of phosphotyrosine (pY) immunoblots. The μg protein values given below each lane indicate the amount of sample protein run in that lane. Lanes 1 to 4 contain incremental amounts of the oxygenated time control (TC) sample. Lanes 5 to 8 are experimental samples taken at the end of 60 minutes of hypoxia (H) with either NES or 4.0 mmol/L αKG/ASP. Each end hypoxia sample is from a separate experiment. Larger amounts of protein were run in the hypoxia lanes to provide signals that are measurable against the most linear portion of the signal range of the control samples. B: Summary of densitometry analysis of the 68- to 75-kd band that corresponds to the location of paxillin. Values are means ± SE for n = 10. *, P < 0.05 versus hypoxia without αKG/ASP.

Immunoblots with phosphorylation state-specific antibodies to paxillin and focal adhesion kinase and after immunoprecipitation with anti-p130^cas or anti-pY. A: Western blots of total cell protein from tubules subjected to 60 minutes of hypoxia followed by 60 minutes of reoxygenation were probed with the indicated phosphorylation state-specific antibodies (top), then the membranes were reprobed (bottom) with the same antibodies used in Figure 5 ▶ , which recognize the proteins irrespective of their phosphorylation state. pY, phosphotyrosine; FAK, focal adhesion kinase; H, sample taken at the end of 60 minutes of hypoxia with NES; R, samples taken from the same flask as the H sample, but obtained after 60 minutes of reoxygenation with either NES or 4.0 mmol/L αKG/MAL; 75′ TC, oxygenated time control at 75 minutes of incubation. Blots shown are all for samples from the same experiment, which is representative of the results of four separate studies analyzed by this approach. B: Representative blots showing the results of: 1) immunoprecipitation (IP) with anti-p130^cas followed by immunoblotting (IB) for pY, then p130^cas; and 2) immunoprecipitation with anti-pY followed by immunoblotting for pY, then p130^cas and paxillin. Experimental protocols and labeling are otherwise the same as for A.

Extent of ATP recovery required for protective effects of supplemental substrates. A: Tubules were subjected to 60 minutes of hypoxia and 60 minutes of reoxygenation with either NES or extra substrates (ES). Four mmol/L concentrations of the supplemental substrates, glutamate plus malate or αKG/MAL, were provided only during reoxygenation. For each condition, tubules were studied with (+) and without (−) AMP (250 μmol/L) in the medium during reoxygenation. Values are means ± SE for n = 4–10. *, Significantly different from corresponding NES/+AMP group; #, significantly different from corresponding ES/−AMP group. Values for control oxygenated tubules (not shown in the figure) at 135 minutes of total incubation corresponding to the end of the reoxygenation period in the experimental flasks were: ATP, 19.2 ± 2.3 nmol/mg protein with AMP and 6.57 ± 0.25 without AMP; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazocarbocyanine iodide (JC-1) 590/530 nm ratio, 4.34 ± 0.06 with AMP and 4.21 ± 0.15 without AMP. B: pY immunoblot for a representative experiment from the series of studies in which αKG/MAL was used as the ES. Lane 1, oxygenated time control (TC); lane 2, hypoxia/reoxygenation with NES, AMP present during reoxygenation (+AMP); lane 3, hypoxia/reoxygenation with ES during reoxygenation, but without AMP (−AMP); lane 4, hypoxia/reoxygenation with ES and AMP present during reoxygenation. The ATP values given for each of the samples were obtained at the end of reoxygenation at the same time as the protein for immunoblotting was collected. pY signals of oxygenated controls were not affected by ES or AMP (not shown).