SPECIAL COMMUNICATION
Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools

Pierre C. Dagher

Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of GTP during ischemia remains unknown. We used LLC-PK proximal tubular cells to measure GTP and ATP changes during anoxia. In 45 min, antimycin A decreased ATP and GTP to 8% and 2% of controls, respectively. Ischemia in vivo resulted in comparable reductions in GTP and ATP. After 2 h of recovery, GTP levels in LLC-PK cells increased to 65% while ATP increased to 29%. We also investigated steady-state models of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26% of control. Selective GTP depletion resulted in significant apoptosis. Selective ATP depletion caused mostly necrosis. These models of ATP or GTP depletion can prove useful in dissecting the relative contribution of the two nucleotides to the ischemic phenotype.

ATP depletion; GTP depletion; mycophenolic acid; alanosine; apoptosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC INJURY TO THE KIDNEY results in profound alterations in morphology and function of the proximal tubular cells. These include reversal of epithelial polarity, changes in actin cytoskeleton dynamics, and disruption of intercellular junctions (9, 20-22). Furthermore, depending on the severity of the injury, cell death in the form of apoptosis or necrosis is observed (15). This complex phenotype of ischemic injury has been best characterized using chemical anoxia models in cell culture (5, 6). These model systems allow the imposition of controlled and graded anoxia followed by recovery (2, 17). They have proved to be powerful tools for dissecting the myriad of biochemical and cell biological events that occur during ischemia.

Of all the potential mediators of ischemic injury, ATP depletion has always played a central role. In fact, ATP depletion has become synonymous with ischemia. This stems from the undisputed function of ATP as the primary energy source of the cell and its equally important role as a signaling molecule. Other mediators of ischemic injury have also been well characterized. These include intracellular pH, calcium, free radicals, and phospholipid products like ceramide (4, 13, 30). They each play an important role in transducing the ischemic insult to generate the final ischemic phenotype.

In contrast, the fate and role of cellular GTP levels during ischemia remains obscure. Viewing the importance of the GTP-GDP molecular switch in a variety of G protein signaling events (19), we hypothesized that changes in the GTP or the GTP/GDP ratio during ischemia are potential factors in the final injury. In fact, the emerging role of the small GTPases in determining cell shape, actin dynamics, proliferation, and apoptosis strongly suggests an important contribution of guanine nucleotides to the changes in cell function during ischemia (7, 8, 25).

We therefore proceeded to quantify the changes in guanine and adenine nucleotides during ischemia and recovery. In addition, because of the observed profound alterations in GTP levels, we established dynamic and steady-state models of selective ATP or GTP depletion and recovery. The effect of selective GTP or ATP depletion was further correlated with the form of cell death. We believe these models will permit a deeper analysis of the functional and morphological changes seen during ischemia than that provided by standard chemical anoxia models.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. A4.8 clones of LLC-PK porcine proximal tubule cells were grown in 100-mm dishes in DMEM with 10% FBS (Sigma Chemical, St. Louis, MO) and 5 mM glucose (referred to as regular medium). For some experiments, depleted media were used consisting of regular media minus amino acids, glucose, and serum. Cells were grown in humidified atmosphere (95% air-5% CO2) at 37°C. Cells were fed 24 h before the experiments and were 2 days into confluence at the time of the experiment. Three washes with warm PBS were performed before starting experimental conditions. After chemical anoxia, only one wash with PBS was performed before recovery to prevent detachment of the monolayer.

Chemicals. Alanosine was a generous gift from the Drug Synthesis and Chemistry Branch of the National Cancer Institute. All other chemicals were from Sigma Chemical. The following final concentrations were used: 50 µg/ml mycophenolic acid (MPA), 1 mM alanosine, 5 mM glucose, 160 µM adenosine, 160 µM guanosine, 0.1 µM antimycin A, 5 mM deoxyglucose, 2 mM pyruvate, 0.1 µg/ml Hoechst 33342, and 1.5 µg/ml propidium iodide.

Nucleotide extraction. At the end of an experimental protocol, the medium was aspirated, and the cells were washed three times with ice-cold PBS. Extraction was done by scraping the cells in 300 µl ice-cold acetonitrile followed by 700 µl cold water (1). The soluble and precipitated fractions were centrifuged at 16,000 g for 10 min at -20°C. The supernatant fraction, kept on ice, was then gassed with N2 for 30 min to evaporate acetonitrile. The pellet was solubilized with 1 N NaOH, and the protein content was analyzed with Coomassie blue assay (Pierce Chemical, Rockford, IL).

HPLC. The column used was a 4-µm Nova-Pak C18 cartridge (100 mm by 8 mm ID), equipped with a radial compression chamber (Waters, Milford, MA). The buffer consisted of 20% acetonitrile, 10 mM ammonium phosphate, and 2 mM PIC Reagent A ion-pairing reagent (Waters) and was run isocratically at 2 ml/min (10). Samples were diluted in half, and the injection volume was 100 µl. A HP Chemstation model 1100 was used (Hewlett-Packard, Wilmington, DE), and the ultraviolet (UV) detector was set at 254 nm. HPLC-grade nucleotide standards were use to calibrate the signals. They were run daily because the retention of the column varied with time. Internal standards were occasionally added to the samples to test recovery. It exceeded 90% for all nucleotides.

Animal studies. Male Sprague-Dawley rats weighing 200-300 g were maintained on standard diet with free access to water. Under pentobarbital anesthesia (5 mg/100 g body wt), the left kidney was subjected to ischemia by occlusion of the renal pedicle for 30 min. Pieces from the cortex of the ischemic kidney and the contralateral control kidney were obtained with a cutting forceps precooled in liquid nitrogen. The tissue was crushed under liquid nitrogen in a mortar with a pestle, and 10-mg samples were resuspended in 600 µl ice-cold acetonitrile and 1,400 µl water. The samples were processed for HPLC as described above.

Fluorescence microscopy. Culture dishes with a central coverslip area suitable for microscopy were used. The adherent cells were visualized after staining directly in the culture dishes. Cells were stained with the vital dye Hoechst 33342, and the morphology of the nucleus was examined for apoptotic features like condensation and fragmentation. Concomitant staining with propidium iodide identified necrotic cells. A Zeiss confocal microscope (LSM 510), equipped with UV and helium lasers, was used. Four to six representative fields (150-250 cells/field) were examined by two observers, one of whom was blinded. Apoptosis and necrosis were expressed as percentage of total cells counted. Because apoptotic cells tend to detach from the monolayer, the culture medium was suctioned and centrifuged, and the pellet was resuspended in a small volume of cold PBS. It was then layered on a slide for microscopic examination, and apoptotic and necrotic cells were counted as before. Control cells showed 0-2% apoptosis and 5-8% necrosis.

Statistics. Results are expressed as means ± SE and were analyzed for significance by paired and unpaired Student's t-test and ANOVA.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In resting LLC-PK cells, the following parameters were measured (values are means ± SE, n = 12): ATP = 46.1 ± 2.5 pmol/µg protein, GTP = 6.1 ± 0.3 pmol/µg protein, ATP/ADP = 7.2 ± 0.6, GTP/GDP = 9.7 ± 0.6, and ATP/GTP = 7.6 ± 0.2. Figure 1 shows representative chromatograms from standards, control cells, chemical anoxia, and recovery. When cells were placed in depleted media and exposed to antimycin A (an inhibitor of oxidative phosphorylation) for 15, 30, or 45 min, GTP decreased at a rate nearly identical to that of ATP. At 45 min, GTP levels were 7.5 ± 2.6%, whereas ATP levels were 1.9 ± 1.5% of controls (Figs. 1C and 2A). To investigate whether GTP is also depleted after ischemia in vivo, we analyzed the nucleotide content of cortices from ischemic and control rat kidneys. Representative chromatograms are shown in Fig. 3. As with chemical anoxia in vitro, 30 min ischemia reduced GTP and ATP to 11 ± 3% and 4 ± 2% of controls, respectively (n = 4).


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Fig. 1.   Effect of anoxia on LLC-PK nucleotide levels: representative chromatograms. A: nucleotide standards in water. B: control LLC-PK cells. C: LLC-PK cells after 45 min of anoxia. D: LLC-PK cells after 2 h recovery after anoxia. 1, IMP + GMP; 2, AMP; 3, GDP; 4, ADP; 5, GTP; and 6, ATP; mAU, milli; absorbance units.



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Fig. 2.   Effect of anoxia on LLC-PK nucleotide levels. Values are means ± SE. A: LLC-PK cells were exposed to 0.1 µM antimycin A in depleted media for 15, 30, and 45 min. B: LLC-PK cells were exposed to 0.1 µM antimycin A in depleted media for 45 min. They were then allowed to recover in regular media in the absence of antimycin A for 2 h. C: LLC-PK cells were exposed to 0.1 µM antimycin A in depleted media for 45 min. They were then bathed in regular media with 5 mM deoxyglucose and 160 µM guanosine for 2 h. D: LLC-PK cells were exposed to 0.1 µM antimycin A in depleted media for 45 min. They were then bathed in depleted media with 5 mM glucose, 2 mM pyruvate, 160 µM adenosine, and 50 µg/ml mycophenolic acid (MPA) for 2 h.



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Fig. 3.   Effect of ischemia on nucleotide levels in rat kidney: representative chromatograms. A: nucleotides extracted from control kidney of Sprague-Dawley rats. B: nucleotides extracted from kidney subjected to 30 min ischemia by renal artery clamp. 1, IMP + GMP; 2, AMP; 3, GDP; 4, ADP; 5, GTP; and 6, ATP.

After 45 min of exposure to antimycin A in depleted media, LLC-PK cells were allowed to recover for 2 h in regular media. As shown in Figs. 1D and 2B, GTP recovered invariably at a greater rate than ATP. After 2 h, GTP reached 65.2 ± 5.4% of control, whereas ATP increased only to 29.0 ± 6.2% of control. After 6 h of recovery, GTP and ATP levels were ~90% and 65%, respectively. Microscopic examination at 6 h showed a mixed picture of necrosis (4%) and apoptosis (6%). Most cells, however, appeared normal (data not shown).

We next attempted selective repletion of GTP after 45 min of antimycin A treatment. This was done by exposing the cells to deoxyglucose (an inhibitor of glycolysis) and guanosine in regular media. As shown in Fig. 2C, GTP recovered to 52.3 ± 10.6% of control after 2 h, whereas ATP remained below 9%. The addition of deoxyglucose alone (without guanosine) inhibited the recovery of both nucleotides. Attempts to achieve selective ATP recovery using MPA (an inhibitor of de novo guanine nucleotide synthesis) with or without adenosine were unsuccessful (data not shown). GTP recovery could not be inhibited. However, when recovery was done in depleted media with MPA, pyruvate, glucose, and adenosine, ATP and GTP recovered at similar rates, reaching ~30% of control at 2 h (Fig. 2D).

Having shown that GTP levels change markedly during the induction and recovery phases of chemical anoxia, we proceeded next to develop steady states of selective ATP or GTP depletion. Figure 4, A and B, shows the condition of moderate selective GTP depletion. It was obtained by exposing the cells to MPA in regular media. GTP decreased to 61.8 ± 4.8% of control within 6 h and remained at this level for at least 24 h. ATP levels remained above 90% of control. The GTP/GDP and ATP/ADP ratios paralleled the levels of GTP and ATP, respectively. At 24 h, microscopic examination revealed 25% of cells with apoptotic features and only 5% with necrosis (Figs. 5 and 6). This high incidence of apoptosis was confirmed by DNA gel electrophoresis showing the typical ladder pattern (data not shown). GTP depletion and cell death could be reversed up to 16 h after MPA addition by bathing the cells in regular media. GTP increased to 80% of control in 4 h, and apoptosis at 24 h was reduced to 10%.


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Fig. 4.   Effect of MPA on LLC-PK nucleotide levels. Values are means ± SE. A: nucleotide content. B: ratio. LLC-PK cells were bathed in regular media with 50 µg/ml MPA. Nucleotides were assayed at 2, 4, 6 and 24 h.



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Fig. 5.   Selective nucleotide depletion and the form of cell death. LLC-PK cells were stained with Hoechst 33342 (blue) and propidium iodide (red) and examined under laser-scanning confocal microscopy. A: control cells showing normal nuclear morphology. B: cells exposed to MPA for 24 h. Typical apoptotic nuclei are seen, with chromatin condensation and fragmentation and no propidium iodide uptake. Only one cell shows mild propidium iodide uptake, indicating secondary necrosis. C: cells exposed to alanosine and guanosine for 24 h. Nuclei show necrotic features with heavy propidium iodide uptake, indicating damaged cell membranes.



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Fig. 6.   The form of cell death during graded selective nucleotide depletion. Values are means ± SE and represent apoptosis or necrosis as % of total cells counted in that condition. Apoptosis and necrosis were identified by microscopy as detailed in METHODS. R-M, regular media with MPA at 24 h (n = 5; 840 cells counted per experiment; *P < 0.005 compared with apoptosis in control, R-Al-G and D-Al-G). R-Aa-M-A, regular media with antimycin A, MPA, and adenosine at 6 h (n = 4; 780 cells counted per experiment; *P < 0.005 compared with apoptosis in control, R-Al-G and D-Al-G). R-Al-G, regular media with alanosine and guanosine at 24 h (n = 5; 900 cells counted per experiment; dagger P < 0.05 compared with necrosis in control, R-M, and D-Al-G). D-Al-G, depleted media with alanosine and guanosine at 6 h (n = 4; 650 cells counted per experiment; Dagger P < 0.01 compared with necrosis in control, R-M, R-Aa-M-Ad, and R-Al-G).

Figure 7, A and B, shows the condition of severe selective GTP depletion. It was achieved by exposing the cells to MPA, antimycin A, and adenosine in regular media. Over 6 h, GTP levels decreased to 26.9 ± 2.2% with ATP remaining at about 70% of control. The GTP/GDP and ATP/ADP ratios paralleled the levels of GTP and ATP, respectively. This condition could not be sustained for 24 h because significant cell death was noted over that time period. At 6-8 h, 20% of cells were apoptotic and 6-8% necrotic (Fig. 6). GTP depletion and cell death could be partially reversed up to 4 h after MPA and antimycin A addition by bathing the cells in regular media.


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Fig. 7.   Effect of MPA and antimycin A on LLC-PK nucleotide levels. Values are means ± SE. A: nucleotide content. B: ratio. LLC-PK cells were bathed in regular media with 50 µg/ml MPA, 0.1 µM antimycin A, and 160 µM adenosine. Nucleotides were assayed at 2, 4, and 6 h.

Figure 8, A and B, shows the condition of moderate selective ATP depletion. This was achieved by exposing the cells to alanosine (an inhibitor of the de novo adenine nucleotide synthetic pathway; Ref. 18) and guanosine in regular media. Over 24 h, ATP levels decreased to 44.5 ± 6.9%, whereas GTP remained near 100% of control. Again, the ATP/ADP and GTP/GDP ratios varied in parallel with ATP and GTP, respectively. At 24 h microscopic examination revealed 1% of cells with apoptotic features and 12% with necrosis (Figs. 5 and 6). ATP depletion and cell death could be reversed up to 12 h after alanosine addition by bathing the cells in regular media. ATP increased to 70% of control after 24 h, and necrosis at 24 h was reduced to 6%.


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Fig. 8.   Effect of alanosine on LLC-PK nucleotide levels. Values are means ± SE. A: nucleotide content. B: ratio. LLC-PK cells were bathed in regular media with 1 mM alanosine. Nucleotides were assayed at 2, 4, 6, and 24 h.

Finally, selective severe ATP depletion over 6 h was induced by exposing the cells to alanosine and guanosine in depleted media (Fig. 9, A and B). ATP decreased to 26.4 ± 1.1%, with GTP remaining near 100% of control. Furthermore, although the ATP/ADP ratio decreased in parallel with ATP, there was a significant decrease in the GTP/GDP ratio, despite the increase in absolute GTP levels. However, at all times, the decrease in the ATP/ADP ratio was greater than that of the GTP/GDP ratio. Again, this condition could not be sustained for 24 h because of significant cell death. At 6-8 h, 2% of cells were apoptotic and more than 30% necrotic (Fig. 6). The addition of insulin and hydrocortisone (to compensate for the absence of growth factors) did not alter the outcome. ATP depletion and cell death could be partially reversed up to 4 h after alanosine addition by bathing the cells in regular media.


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Fig. 9.   Effect of alanosine and depleted media on LLC-PK nucleotide levels. Values are means ± SE. A: nucleotide content. B: ratio. LLC-PK cells were bathed in depleted media media with 1 mM alanosine and 160 µM guanosine. Nucleotides were assayed at 2, 4, and 6 h.

Figure 10 shows the value of the ATP/GTP ratio obtained at 6 h in the conditions described above. In the anoxia/recovery models, ATP/GTP ranged from 20% to 100% of control (Fig. 10A). This spectrum was broadened in the selective depletion models, ranging from 25% to 245% of control (Fig. 10B). The significance of the ATP/GTP ratio is discussed below.


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Fig. 10.   The spectrum of ATP/GTP ratios. Values are means ± SE and represent the ATP/GTP ratio obtained from the anoxia/recovery experiments (A) and the 6-h time point from the various conditions of selective depletion (B). R-Aa-M-P, regular media with antimycin A, MPA, and adenosine. R-M, regular media with MPA; R-Al-G, regular media with alanosine and guanosine; D-Al-G, depleted media with alanosine and guanosine.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we have shown that, both in vivo and in vitro, GTP levels fall dramatically during ischemia or chemical anoxia. These results point to GTP as an important variable that could modulate the effects traditionally ascribed to ATP. A study by Doctor et al. (6) has previously shown GTP to decrease only to 50-75% of control after 30 min of anoxia. This contrasts with the more severe reduction (<10% of control) reported in this study. Possible reasons for this difference include their use of a HPLC protocol in which GTP coelutes in a split peak with CMP (28). Because CMP is likely to increase during anoxia, this could lead to overestimation of GTP. In addition, nucleotides are notoriously difficult to resolve with reverse-phase protocols in the absence of ion pairing (10). Furthermore, perchloric acid extraction can interfere with peak resolution due to residual perchlorate ions, a problem avoided by using acetonitrile extraction (1, 29). Our results clearly show that both in vivo and in vitro, GTP is significantly affected by ischemia or anoxia.

A surprising finding was that GTP recovered faster than ATP after chemical anoxia. However, the rate of ATP recovery reported in the literature is very variable. Factors that could affect it include the clone of cells used, the duration of anoxia, the inhibitor used, and the degree of washing performed between anoxia and recovery (5). The rate of recovery in our hands is similar to that reported by Doctor et al. (6) when they used antimycin A. When rotenone was used, they observed enhanced ATP recovery (close to 100% at 1 h).

Our model of selective GTP recovery after chemical anoxia should prove useful in dissecting a role for GTP from contributions of ATP recovery. For example, cytoskeletal changes after anoxia have been recently linked to Rho GTPase activity (25). Thus GTP levels during this phase could be important in modulating Rho activity. Our failure to induce a state of selective ATP recovery could reflect activation of nucleoside mono- and diphosphate kinase. This enzyme equilibrates guanine and adenine di- and triphosphate pools. As most guanines are in the monophosphate form after anoxia, this enzyme could utilize ATP during recovery to regenerate GTP form GMP and GDP. Addition of MPA cannot prevent this, as it inhibits the step from IMP to GMP. Inhibitors of nucleoside phosphate kinase are not readily available but could permit the realization of this condition (27).

Metabolic inhibitors like MPA or alanosine have been used before to selectively deplete nucleotide pools. However, these studies address issues relating the role of specific nucleotides in controlling insulin secretion, cell proliferation, or lymphocyte function (18, 23, 26). To our knowledge, this study is unique in being specifically tailored to model epithelial ischemia in vitro. It does so by providing both dynamic and steady-state conditions in which one of the nucleotides is selectively altered.

The steady-state conditions of graded selective ATP or GTP depletion will permit the evaluation of the role of these two nucleotides in diverse phenomena seen during ischemia. Issues such as cytoskeletal rearrangement, Golgi trafficking, cell polarity, and cell death can now be related to one or both of the two nucleotides. In fact, the functional significance of these models is underscored by our finding that apoptosis is induced specifically by GTP depletion. Other studies have ascribed apoptosis to moderate ATP depletion (15). However, GTP was not measured in these studies, and the protocols used to deplete ATP actually cause both ATP and GTP depletion. Our results show only necrosis when ATP is selectively depleted. Thus GTP levels seem to be important in modulating the form of cell death observed during anoxia. The mechanism of GTP-induced apoptosis was not investigated in this study but has been reported by others (14, 26)

The ATP/GTP ratio is an important parameter in cell physiology. As discussed by Liu et al. (16), it is at the heart of the coordination between signals involving GTP hydrolysis or ATP-mediated phosphotransfer. It is a remarkably constant ratio and shows little variation between different cell lines (3, 12). It deviates from normal only under conditions of severe stress or neoplastic transformation. Its role in the delicate integration of signaling cascades is underscored by the occurrence of variants to the normal flow of information. These include protein kinases that can use GTP and small GTPases that can hydrolyze ATP (11, 24). The large spectrum of ATP/GTP values achieved with our models will permit the evaluation of the role of this ratio in epithelial biology.

In summary, we have shown that GTP levels change dramatically during chemical anoxia and recovery. This points to GTP as a potential important factor in determining the ischemic phenotype. We have further examined models of selective GTP or ATP manipulations of graded severity. They revealed that selective GTP depletion and not ATP depletion is a cause of apoptosis in LLC-PK cells. Therefore, these models have the potential to allow the dissection of the relative roles of ATP and GTP in epithelial function. This could be of importance in designing therapeutic strategies, because it would permit specific targeting of the responsible nucleotide.


    ACKNOWLEDGEMENTS

I thank Melanie Hosford, Zoya Plotkin, and Rubin Sandoval for technical assistance and Bruce Molitoris for critical reading of the manuscript. I am very grateful to Bruce Molitoris for advice and support throughout this project.


    FOOTNOTES

This work was supported in part by a grant from the National Kidney Foundation-Indiana.

Address for reprint requests and other correspondence: P. C. Dagher, Dept. of Medicine, Div. of Nephrology, Indiana Univ. School of Medicine, Fesler Hall 115, 1120 South Dr., Indianapolis, IN 46202 (E-mail: pdaghe2{at}iupui.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 January 1999; accepted in final form 17 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Au, JL, Su MH, and Wientjes MG. Extraction of intracellular nucleosides and nucleotides with acetonitrile. Clin Chem 35: 48-51, 1989[Abstract/Free Full Text].

2.   Bacallao, R, Garfinkel A, Monke S, Zampighi G, and Mandel LJ. ATP depletion: a novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton. J Cell Sci 107: 3301-3313, 1994[Abstract/Free Full Text].

3.   Balzarini, J, Karlsson A, Wang L, Bohman C, Horska K, Votruba I, Fridland A, Van Aerschot A, Herdewijn P, and De Clercq E. Eicar (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide). A novel potent inhibitor of inosinate dehydrogenase activity and guanylate biosynthesis. J Biol Chem 268: 24591-24598, 1993[Abstract/Free Full Text].

4.   Bor, MV, Durmus O, Bilgihan A, Cevik C, and Turkozkan N. The beneficial effect of 2'-deoxycoformycin in renal ischemia-reperfusion is mediated both by preservation of tissue ATP and inhibition of lipid peroxidation. Int J Clin Lab Res 29: 75-79, 1999[ISI][Medline].

5.   Canfield, PE, Geerdes AM, and Molitoris BA. Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells. Am J Physiol Renal Fluid Electrolyte Physiol 261: F1038-F1045, 1991[Abstract/Free Full Text].

6.   Doctor, RB, Bacallao R, and Mandel LJ. Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures. Am J Physiol Cell Physiol 266: C1803-C1811, 1994[Abstract/Free Full Text].

7.   Exton, JH. Small GTPases minireview series. J Biol Chem 273: 19923, 1998[Free Full Text].

8.   Gomez, J, Martinez AC, Gonzalez A, and Rebollo A. Dual role of Ras and Rho proteins: at the cutting edge of life and death. Immunol Cell Biol 76: 125-134, 1998[ISI][Medline].

9.   Gopalakrishnan, S, Raman N, Atkinson SJ, and Marrs JA. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol Cell Physiol 275: C798-C809, 1998[Abstract].

10.   Grune, T, Siems W, Gerber G, Tikhonov YV, Pimenov AM, and Toguzov RT. Changes of nucleotide patterns in liver, muscle and blood during the growth of Ehrlich ascites cells: application of the reversed-phase and ion-pair reversed-phase high-performance liquid chromatography with radial compression column. J Chromatogr 563: 53-61, 1991[Medline].

11.   Hide, G, Graham T, Buchanan N, Tait A, and Keith K. Trypanosoma brucei: characterization of protein kinases that are capable of autophosphorylation in vitro. Parasitology 108: 161-166, 1994[ISI][Medline].

12.   Jayaram, HN, Zhen W, and Gharehbaghi K. Biochemical consequences of resistance to tiazofurin in human myelogenous leukemic K562 cells. Cancer Res 53: 2344-2348, 1993[Abstract].

13.   Kristian, T, and Siesjo BK. Calcium in ischemic cell death. Stroke 29: 705-718, 1998[Abstract/Free Full Text].

14.   Li, G, Segu VB, Rabaglia ME, Luo RH, Kowluru A, and Metz SA. Prolonged depletion of guanosine triphosphate induces death of insulin-secreting cells by apoptosis. Endocrinology 139: 3752-3762, 1998[Abstract/Free Full Text].

15.   Lieberthal, W, Menza SA, and Levine JS. Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells. Am J Physiol Renal Physiol 274: F315-F327, 1998[Abstract/Free Full Text].

16.   Liu, Y, Bohn SA, and Sherley JL. Inosine-5'-monophosphate dehydrogenase is a rate-determining factor for p53-dependent growth regulation. Mol Biol Cell 9: 15-28, 1998[Abstract/Free Full Text].

17.   Mandel, LJ, Doctor RB, and Bacallao R. ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K+-ATPase and E-cadherin. J Cell Sci 107: 3315-3324, 1994[Abstract/Free Full Text].

18.   Meredith, M, Rabaglia M, and Metz S. Cytosolic biosynthesis of GTP and ATP in normal rat pancreatic islets. Biochim Biophys Acta 1266: 16-22, 1995[ISI][Medline].

19.   Millman, JS, and Andrews DW. Switching the model: a concerted mechanism for GTPases in protein targeting. Cell 89: 673-676, 1997[ISI][Medline].

20.   Molitoris, BA, Leiser J, and Wagner MC. Role of the actin cytoskeleton in ischemia-induced cell injury and repair. Pediatr Nephrol 11: 761-767, 1997[ISI][Medline].

21.   Molitoris, BA, and Wagner MC. Surface membrane polarity of proximal tubular cells: alterations as a basis for malfunction. Kidney Int 49: 1592-1597, 1996[ISI][Medline].

22.   Nigam, S, Weston CE, Liu CH, and Simon EE. The actin cytoskeleton and integrin expression in the recovery of cell adhesion after oxidant stress to a proximal tubule cell line (JTC-12). J Am Soc Nephrol 9: 1787-1797, 1998[Abstract].

23.   Olah, E, Csokay B, Prajda N, Kote-Jarai Z, Yeh YA, and Weber G. Molecular mechanisms in the antiproliferative action of taxol and tiazofurin. Anticancer Res 16: 2469-2477, 1996[ISI][Medline].

24.   Onozawa, T, Danjoh I, and Fujiyama A. Biochemical similarity of Schizosaccharomyces pombe ras1 protein with RAS2 protein of Saccharomyces cervisiae. Yeast 11: 801-808, 1995[ISI][Medline].

25.   Raman, N, and Atkinson SJ. Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery. Am J Physiol Cell Physiol 276: C1312-C1324, 1999[Abstract/Free Full Text].

26.   Savini, F, Rucci C, Messina E, Quaratino CP, Scarpa S, Vasaturo F, Modesti A, and Giacomello A. Differentiating and biochemical effects of a reduction of intracellular GTP levels induced by mycophenolic acid (MPA) in human neuroblastoma (NB) cell lines. Adv Exp Med Biol 431: 443-446, 1998[ISI][Medline].

27.   Schneider, B, Xu YW, Janin J, Veron M, and Deville-Bonne D. 3'-Phosphorylated nucleotides are tight binding inhibitors of nucleoside diphosphate kinase activity. J Biol Chem 273: 28773-28778, 1998[Abstract/Free Full Text].

28.   Taylor, MW, Hershey HV, Levine RA, Coy K, and Olivelle S. Improved method of resolving nucleotides by reversed-phase high-performance liquid chromatography. J Chromatogr 219: 133-139, 1981[Medline].

29.   van Doorn, JE, Goormachtig PF, and de Haan A. Influence of perchloric acid on ion-pair high-performance liquid chromatography of nucleotides. J Chromatogr 496: 441-449, 1989[Medline].

30.   Zager, RA, Iwata M, Conrad DS, Burkhart KM, and Igarashi Y. Altered ceramide and sphingosine expression during the induction phase of ischemic acute renal failure. Kidney Int 52: 60-70, 1997[ISI][Medline].


Am J Physiol Cell Physiol 279(4):C1270-C1277
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