Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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 |
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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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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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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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