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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

AMP deaminase (AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte-specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3-deficient [A3(−/−)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC-AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase (PK) double-deficient [PKA(−/−,−/−)] mice by mating A3(−/−) mice with CBA-Pk-1slc/Pk-1slc mice [PK(−/−)], a spontaneous PK-deficient strain showing hemolytic anemia. In PKA(−/−,−/−) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(−/−) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(−/−) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(−/−) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

AMP deaminase (AMPD) converts AMP to IMP, and plays an important role in energy charge stabilization and nucleotide metabolism (Qiu et al. 2000; Sabina & Holmes 2001; 1992; Haas & Sabina 2003). AMPD becomes activated to remove accumulated AMP in an adenylate pool during periods of energy imbalance in most tissues, providing additional ATP to these energy-challenged cells. In mammals, AMPD has tissue-specific isoforms encoded by 3 genes (Sermsuvitayawong et al. 1997; Hellsten et al. 1999; Szydlowska et al. 2004): skeletal muscle-type (AMPD1), liver-type (AMPD2), and erythrocyte (red blood cell, RBC)-type (AMPD3). AMPD1 is nearly exclusively expressed in skeletal muscles, whereas AMPD2 is rather widely expressed in nonmuscle tissues and cells (Ogasawara et al. 1974). AMPD3 was originally identified in RBCs and heart tissue, and then later found to be expressed in the liver, kidney cortex, brain, and heart, as well as other tissues (Ogasawara et al. 1974; Wang et al. 1997). Deficiencies of the skeletal muscle-type (AMPD1) and RBC-type (AMPD3) have been reported in humans (Morisaki et al. 1992; Yamada et al. 1994a,b). Individuals with AMPD1 deficiency become easily fatigued and show post-exertional muscle aches, cramps, and pain, although the precise cellular mechanisms of AMPD for regulating energy balance are not clearly understood.

AMPD3 is the sole isoform of AMPD expressed in RBCs (Ogasawara et al. 1982). Human RBC AMPD3 deficiency has been reported (Ogasawara et al. 1984), although affected patients do not exhibit any clinically relevant symptoms, while an increase in the level of steady-state ATP in erythrocytes has been observed (Ogasawara et al. 1987). The function of AMPD3 in RBCs may be different from that in other tissues and cells, because of a lack of oxidative ATP production. It was recently reported that AMPD3 plays a role in determining the correct energy state of RBCs under oxidative stress and activation of AMP deaminase induced by oxidative stress (Qiu et al. 2000; Tavazzi et al. 2000). However, the physiological and pathological roles of AMPD3 deficiency remain largely unknown.

Pyruvate kinase (PK) is a key glycolytic enzyme that catalyzes transphosphorylation from phosphoenolpyruvate (PEP) to ADP, yielding pyruvate and ATP. PK exists as a homotetramer in nearly all organisms, with 4 PK isoenzymes (M1, M2, L, and R) identified in mammals and expressed in specific tissues (Imamura & Tanaka 1972). PK plays a central role in RBC metabolism, as it catalyzes 1 of the 2 major steps of ATP production in cells. However, because mature RBCs lack mitochondria, they are absolutely dependent on glycolysis for maintaining cell function. RBC pyruvate kinase deficiency, the most frequently encountered enzyme abnormality of the glycolytic pathway, causes hereditary nonspherocytic hemolytic anemia (Miwa et al. 1993). To date, more than 150 different mutations of the PK-LR gene have been identified to cause PK deficiency and no specific therapy for PK deficiency is presently available. Thus, the primary treatment for PK deficiency is based on supportive measures, such as RBC transfusion or splenectomy, although gene transfer studies of human PK cDNA in hematopoietic stem cells of lethally irradiated mice have been presented (Tani et al. 1994). Nevertheless, the role of nucleotide metabolism in PK-deficient RBCs remains unclear, and the effects of therapy targeting ATP levels related to anemia in PK deficiency have not been extensively examined.

To test the physiological role of AMPD3, we established a line of AMPD3-deficient [A3(−/−)] mice that showed elevated levels of ATP in RBCs, as also reported in human AMPD3 deficiency. There was no remarkable phenotype seen in observations of the A3(−/−) mice raised under normal conditions. Next, we examined the therapeutic effects of altered ATP levels on anemia due to PK deficiency by establishing AMPD3 and PK double-deficient [PKA(−/−,−/−)] mice by mating A3(−/−) with CBA-Pk-Islc/Pk-1slc [PK(−/−)] mice, the latter of which spontaneously develop PK deficiency (Tsujino et al. 1998). In PKA(−/−,−/−) mice, the level of ATP in RBCs was significant higher than that in those of PK(−/−) mice. However, anemia, splenomegaly, and increment of reticulocytes in PKA(−/−,−/−) mice were not improved. Rather, osmotic fragility test results revealed that RBCs obtained from A3(−/−) mice after fasting were more susceptible to a low osmotic condition than those from the control. Together, these findings suggest that AMPD3 deficiency is associated with an increased level of ATP in RBCs, although there is no therapeutic effect on anemia caused by PK deficiency, which was shown to increase the osmotic fragility of RBCs in a fasting condition.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Confirmation of disruption of AMPD3 gene

We detected the AMPD3 gene by PCR and Southern blotting analyses to confirm AMPD3 knockout (Fig. 1B). In Southern blotting findings, knockout mice showed bands of 2.6 and 0.6 kb with 3′ and 5′ probes, respectively (Fig. 1B). AMPD activity was assessed in RBCs as well as several types of tissues, including liver, kidney, and heart samples (Table 1). That activity was found to be undetectable or extremely low in the RBCs and hearts of A3(−/−) mice, while significant decreases were also observed in liver, kidney and heart tissues of those mice. In skeletal muscle, AMPD activity in A3(−/−) mice showed slight decrease compared with that in wild-type mice (280 mU/mg protein vs 430 mU/mg protein). These results confirmed that an AMPD3-deficient mouse line was successfully established and that AMPD3 was the only gene for AMP deaminase expressed in the RBCs of these mice.

image

Figure 1. Generation of AMPD3 knockout mice and confirmation of disruption of AMPD3 gene. (A) Gene targeting strategy. (B) Southern blot analyses of tail DNA from offspring derived from heterozygous intercrossing.

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Table 1. Total AMPD activity in various tissues (mU/mg protein)
 ErythrocytesLiverKidneyHeart
  1. 12-week-old male mice (n = 6). Values are shown as the mean ± SE.

  2. a

    P < 0.05 (vs WT).

WT5.24 ± 0.419.2 ± 1.041.5 ± 1.061.4 ± 3.9
A3(−/−)a0.00a13.5 ± 1.0a14.4 ± 0.60a2.90 ± 0.2

General observations of A3(−/−) mice

There were no remarkable phenotype differences between A3(−/−) and wild-type mice kept in a controlled SPF environment with a 12-h light–dark cycle, constant temperature (25 °C), and free access to food (CE-2, CLEAR) and water. The experimental mice were born with the expected Mendelian ratio and fertile, and appeared indistinguishable from their wild-type littermates, with no significant differences in body weight, body composition, food intake, and life span (data not shown). HE staining of liver, kidney, lung, brain, heart, spleen, and stomach tissue sections also revealed no significant differences (data not shown).

The morphology of the RBCs was observed using Giemsa staining, with no obvious difference seen between those from A3(−/−) and wild-type mice. There were also no significant differences found for Hb, RBC, WBC, and MCH levels between the AMPD3-deficient and wild-type mice, whereas there was a limited but significant elevation (nearly 5%) of MCV in the A3 (−/−) mice (Table 2).

Table 2. Complete blood counts
 WTA3(−/−)PK(−/−)PKA(−/−, −/−)
  1. 12-week-old male mice (n = 6). Values are shown as the mean ± SE.

  2. a

    P < 0.05 (vs WT).

WBCs (×103/µL)8.6 ± 1.27.4 ± 1.17.3 ± 0.78.3 ± 0.9
RBCs (×106/µL)8.9 ± 0.28.6 ± 0.25.5 ± 0.2a5.3 ± 0.2a
Hb (g/dL)14.1 ± 0.213.7 ± 0.29.7 ± 0.4a9.1 ± 0.2a
Ht (%)46.6 ± 1.146.8 ± 1.036.3 ± 0.3a34.5 ± 0.4a
MCV (fL)52.1 ± 1.154.6 ± 0.9a65.7 ± 0.3a65.5 ± 2.1a
MCH (pg)15.8 ± 0.216.1 ± 0.217.5 ± 0.2a17.2 ± 0.7a
MCHC (%)30.3 ± 0.629.3 ± 0.426.7 ± 0.2a26.3 ± 0.2a
Ret(%)2.3 ± 0.32.1 ± 0.150.7 ± 3.5a47.7 ± 2.6a

A3 (−/−) mice exhibit elevated AMP, ADP, and ATP levels in RBCs

Human RBC AMPD deficiency is known to be accompanied with an increased level of ATP in RBCs. To determine whether murine AMPD3 deficiency is related to the change of nucleotide levels in RBCs, the levels of AMP, ADP, ATP, and GTP in RBCs of A3(−/−) mice were investigated using HPLC. ATP, ADP, and AMP levels in RBCs of A3(−/−) mice were elevated by 2.6-, 2.0-, and 3.5-fold as compared to the control (Table 3), although a significant decrease in GTP level was observed in RBCs from A3(−/−) mice. These results indicated a significant change in nucleotide levels in RBCs caused by AMPD3 deficiency, suggesting the usefulness of A3(−/−) mice as a murine model for human RBC AMPD deficiency.

Table 3. Nucleotide levels in RBCs (nmol/mg Hb)
  A3PKPKAPKAPKAPKPKA
 WT(−/−)(−/−)(−/−, −/−)(−/−,+/−)(+/−,−/−)(+/−)(+/−,+/−)
  1. 12-week-old male mice (n = 6). Values are shown as the mean ± SE.

  2. a

    P < 0.05 (vs WT).

  3. PKA(−/−, +/−): PK(−/−)A3(+/−); PKA(+/−,−/−): PK(+/−)A3(−/−); PKA(+/−, +/−): PK(+/−)A3(+/−).

ATP13.335.4a11.917.5a10.3a13.05.1a3.8a
±1.5±2.1±0.6±1.6±0.4±0.6±0.4±0.2
ADP3.97.7a3.54.1a3.35.8a2.2a2.9a
±0.7±0.7±0.2±0.5±0.1±0.7±0.0±0.1
AMP0.41.4a0.51.4a0.61.0a0.60.7a
±0.1±0.4±0.0±0.0±0.0±0.1±0.1±0.0
GTP2.40.5a2.62.0a2.30.2a1.0a0.3a
±0.1±0.0±0.1±0.2±0.2±0.1±0.2±0.1

No improvement of anemia was observed in association with PK deficiency when AMPD3 deficiency was introduced

PKA(−/−,−/−) mice were born with the expected Mendelian ratio and were fertile, whereas they were visually indistinguishable from their PK(−/−) littermates. In PKA(−/−,−/−) mice, AMPD activity in RBCs was undetectable, the same as in A3(−/−) mice (data not shown). A complete blood count was performed and PKA(−/−,−/−) mice did show anemia as seen in PK(−/−) mice (Table 2). Increased reticulocyte count and splenomegaly were observed in PKA(−/−,−/−) mice, as well as in PK(−/−) mice (Fig. 2). The anemic conditions observed in PK(−/−) and PKA(−/−,−/−) mice were similar regardless of age (comparisons made at 2, 12, 20, and 32 weeks old) (data not shown).

image

Figure 2. Number of reticulocytes in blood and spleen size. (A) Peripheral blood reticulocytes from wild-type (WT), A3(−/−), PK(−/−), and PKA(−/−,−/−) mice after staining with 1% methylene blue. (B) Splenomegaly was found in PK(−/−) and PKA(−/−,−/−) mice, whereas spleen sizes were normal in the wild-type (WT) and A3(−/−) mice.

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Anemic conditions were also compared among PK(+/−), PKA(+/−,−/−), PKA(+/−,+/−), and PKA (−/−,+/−) mice. No anemia or splenomegaly was found in PK(+/−), PKA(+/−,−/−), or PKA (+/−,+/−) mice, whereas PKA(−/−,+/−) mice exhibited both to the same extent as seen in PK(−/−) and PKA(−/−,−/-) mice (data not shown).

ATP levels significantly increased in RBCs of PKA (−/−,−/−) mice

Next, we investigated nucleotide levels in RBCs by determining the contents of ATP, ADP, AMP, and GTP in those of wild-type, A3(−/−), PK(−/−), PK(+/−), PKA(−/−,−/−), PKA(+/−,−/−), PKA(+/−,+/−), and PKA(−/−,+/−) mice (Table 3). In PKA(−/−,−/−) mice, the ATP level was found to be 1.5 times higher than that in PK(−/−) mice, although it was not as high as the level in A3(−/−) mice. Furthermore, PKA(−/−,−/−) mice showed higher (1.2- and 2.8-fold, respectively) AMP and ADP levels than found in the PK(−/−) mice (Table 3). Interestingly, a significant increase in ATP, ADP, and AMP levels was found in PKA(+/−,−/−) mice as compared to PK (+/−) mice.

A3 (−/−) mice exhibited osmotic fragility after fasting but no hemolysis

Results of osmotic fragility testing revealed that RBCs in A3(−/−) mice after 24 h of fasting were more fragile under a low osmotic condition than those in the control mice. However, that fragility did not worsen after longer fasting periods of 48 and 72 h (Fig. 3). In contrast, there was no difference in the osmotic fragility between PKA(−/−, −/−) and PK(−/−) mice (Fig. S1 in Supporting Information).

image

Figure 3. Osmotic fragility in AMPD3-deficient RBCs before and after fasting. We tested blood samples obtained from wild-type (WT) and A3(−/−)mice (males, 12 weeks old, n = 9–20). Values are shown as the mean ± SE. *P < 0.05

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We also examined whether hemolysis occurs under a fasting condition. The reticulocyte count was not increased in A3(−/−) mice before and after fasting for 72 h, with no hemoglobinuria found under the fasting condition. In liver and spleen tissue sections, osmotic fragility of RBCs in A3(−/−) mice was seen, although there was no evidence for hemolysis or hemolytic anemia under the fasting condition. A complete blood count examination revealed a slightly elevated MCV level in these mice, although no change was found between before and after fasting for any duration (Table 4). There were also no morphological changes of RBCs in A3(−/−) mice associated with fasting.

Table 4. Complete blood count after fasting
 24 h48 h72 h
WTA3(−/−)WTA3(−/−)WTA3(−/−)
  1. 12-week-old male mice (n = 6). Values are shown as the mean ± SE.

  2. a

    P < 0.05 [WT vs A3(−/−)].

WBCs (×103/µL)6.7 ± 1.25.1 ± 0.83.7 ± 0.83.8 ± 1.31.8 ± 0.31.6 ± 0.1
RBCs (×106/µL)8.9 ± 0.08.9 ± 0.19.5 ± 0.29.3 ± 0.29.5 ± 0.49.1 ± 0.2
Hb (g/dL)14.1 ± 0.314.6 ± 0.215.0 ± 0.315.0 ± 0.315.0 ± 0.714.8 ± 0.2
Ht (%)46.4 ± 0.950.1 ± 0.7a50.3 ± 1.252.2 ± 1.249.9 ± 2.250.1 ± 0.8
MCV (fL)52.6 ± 0.656.0 ± 0.7a52.9 ± 0.856.0 ± 1.2a52.8 ± 1.055.3 ± +1.0a
MCH (pg)15.8 ± 0.316.4 ± 0.215.8 ± 0.316.3 ± 0.215.9 ± 0.316.4 ± 0.2
MCHC (%)30.3 ± 0.329.1 ± 0.329.9 ± 0.328.8 ± 0.5a30.0 ± 0.229.6 ± 0.3

Nucleotide level markedly decreased after fasting regardless of genotype

To investigate changes in nucleotide metabolism after fasting, adenine nucleotide (ATP, ADP, and AMP) levels in RBCs were determined. Those were decreased markedly in both wild-type and A3(−/−) mice after fasting for 24–72 h, although each was higher in the A3(−/−) mice at all time points (Table 5). In addition, the level of GTP in RBCs was decreased after fasting in both A3(−/−) and wild-type mice, although those were significantly lower in A3(−/−) mice at all time points.

Table 5. Nucleotide levels in RBCs after fasting (nmol/mg Hb)
 24 h48 h72 h
WTA3(−/−)WTA3(−/−)WTA3(−/−)
  1. 12-week-old male mice (n = 6). Values are shown as the mean ± SE.

  2. a

    P < 0.05 [WT vs A3(−/−)].

ATP9.2 + 0.725.6 ± 3.3a8.3 ± 0.223.6 ± 1.2a7.6 ± 0.420.3 ± 3.4a
ADP1.8 ± 0.33.1 ± 0.2a1.4 ± 0.52.2 ± 0.3a1.6 ± 0.22.3 ± 0.2a
AMP14.1 ± 0.314.6 ± 0.215.0 ± 0.315.0 ± 0.315.0 ± 0.714.8 ± 0.2
GTP1.1 ± 0.10.2 ± 0.1a1.2 ± 0.10.3 ± 0.1a1.3 ± 0.20.2 ± 0.1a

The levels of serum glucose were also measured before and after fasting for 24–72 h. Those were markedly low in the A3, PK, and PKA genotypes, with no significant difference seen between A3(−/−) and wild-type mice, or between PK(−/−) and PKA(−/−, −/−) mice at any time point (data not shown).

AMPD3-deficient RBCs exhibited higher PRPPS activity

Patients with phosphoribosylpyrophosphate (PRPP) synthetase (PRPPS) deficiency have been reported to have elevated ATP in RBCs and exhibit hemolytic anemia (Valentine et al. 1972), thus PRPPS activity in RBCs was determined to investigate whether AMPD deficiency has an effect. PRPPS activity was significantly higher in the RBCs of A3(−/−) mice as compared to those of the wild type (Fig. 4). Therefore, AMPD3 deficiency may affect nucleotide levels in RBCs, whereas its effect on RBC functions might occur in a manner separate from PRPPS deficiency.

image

Figure 4. PRPPS activity in AMPD3-deficient RBCs. We tested blood samples obtained from wild-type (WT) and A3(−/−) mice (males, 12 weeks old, n = 6). Values are shown as the mean ± SE. *P < 0.05

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

AMPD is widely distributed throughout mammalian tissues and cells, and thought to play important roles in regulating adenylate energy charge, although expression levels vary in different tissues. AMPD3 is a unique type of AMPD expressed in RBCs. Inherited AMPD3 deficiency occurs in humans (Ogasawara et al. 1987), who are asymptomatic and characterized by elevated levels of ATP in RBCs. AMPD3 has been implicated in a number of physiological processes, although its precise function is not clearly understood. To further investigate its function, we first established a murine model line with AMPD3 deficiency, A3(−/−) mice, which have no AMPD activity, along with high levels of AMP, ADP, and ATP in RBCs. Furthermore, A3(−/−) mice do not show any apparent phenotype differences with wild-type mice, including body weight, life span, blood biochemical tests, and blood counts. These characteristics are the same as reported in humans with AMPD3 deficiency, including the significant increase of adenine nucleotide (ATP, ADP, and AMP) levels in RBCs. However, the level of MCV, an indicator of RBC size, was found to be slightly increased in A3(−/−) mice. Based on these results, we speculated that A3(−/−) mice could function well as a model of human AMPD3 deficiency, with elevated ATP and altered nucleotide levels in RBCs, and without a physiologically harmful phenotype. We also consider that this model will be useful to investigate the role of AMPD3 in other tissues, such as those of the heart, brain, kidney, muscle, and liver.

We also investigated the physiological relevance of increased ATP level in AMPD3-deficient RBCs and whether such an increase would improve anemia in a hemolytic anemia model of PK deficiency, in which ATP production is decreased. Although AMPD3-deficient RBCs did not show any apparent physiological changes in RBCs, we hypothesized that AMPD3 deficiency could provide beneficial effects on RBCs with glycolytic defect such as PK deficiency. PKA(−/−, −/−) mice, with both PK and AMPD3 deficiency in RBCs, had significantly increased (1.5-fold) ATP in RBCs. However, there were no improvements found in their anemic condition or hemolysis. These findings indicate that both AMPD3 and PK effectively regulate the ATP pool in RBCs, whereas PK plays a predominant role in RBC function.

As indicated in previous reports (Miwa et al. 1993), ATP is produced almost exclusively by oxidative phosphorylation in reticulocytes in RBCs with severe PK deficiency. Therefore, the increased ATP levels may be explained by increased reticulocytes in PK (−/−) mice. In contrast, ATP levels were also increased in PK (+/−) mice, although they did not exhibit anemia. Therefore, ATP level may not be the only determinant to define RBC function and an independent PK pathway in PK (−/−) mice may exist, as previous reports have noted (Tsujino et al. 1998). Although no anemia was observed in PK (+/−) and PKA (+/−, +/−) mice, they only had half of the ATP content in RBCs as compared to wild-type mice, which suggests that such a reduced ATP content is not enough to cause anemia in these mice. Although PK plays a central role in ATP production by catalyzing 1 of the 2 major steps of ATP production in RBCs and ATP content in RBCs in increased by AMPD3 deficiency, other mechanisms rather than regulation of ATP levels in RBCs are important for their function. These issues remain to be clarified.

In addition, we found that AMPD3 deficiency caused osmotic fragility of RBCs under a fasting condition, showing that AMPD3 is necessary to maintain RBC function while fasting. Although inherited AMPD3 deficiency in humans is reported to be asymptomatic, RBC functions were not evaluated under a fasting condition in that study. We did not find evidence of hemolysis or hemolytic anemia in AMPD3 deficiency, thus RBCs in AMPD3-deficient mice have functional changes. However, RBC size was significantly increased in A3(−/−) mice after fasting, in contrast to the wild type.

ATP level in RBCs is important to maintain cell functions. A number of reports have shown that low a ATP level in RBCs causes osmotic fragility and hemolytic anemia (de Gruchy & R Grimes 1972). Furthermore, hyperglycemia has been reported to induce membrane lipid peroxidation and osmotic fragility in RBCs (Jain 1989). In the present study, ATP, ADP, and AMP levels in RBCs of A3(−/−) mice were found to be decreased under the fasting condition, which caused osmotic fragility of RBCs, although they remained at higher levels than those in wild-type mice. Although precise mechanisms remained to be clarified, functions of Na+-K+ pump or membrane components in RBCs of A3(−/−) mice might be deteriorated by changes of the nucleotide levels after fasting because the mean RBC volume (MCV) were shown to be increased. Accordingly, osmotic fragility was found even in the presence of a high ATP content in RBCs after fasting.

PRPPS is an important enzyme in the synthesis of phosphoribosylpyrophosphate and AMP from ribose 5-phosphate (Duley et al. 2011). PRPPS deficiency has been reported to increase ATP in RBCs and cause hemolytic anemia (Valentine et al. 1972), although the mechanism remains unclear. Therefore, we determined PRPPS activity in AMPD3-deficient RBCs to elucidate the relationship between AMPD3 and PRPPS and found that PRPPS activity was doubled in AMPD3-deficient RBCs. Although this result does not support a common pathway for AMPD3 deficiency and PRPPS deficiency in RBC dysfunction, it provides evidence that AMPD3 deficiency might cause changes in the purine de novo synthesis pathway in RBCs via changes in PRPPS activity. The precise mechanism of the osmotic fragility of RBCs in AMPD3 deficiency requires further exploration.

In summary, we established A3(−/−) mice as a model for investigating human AMPD3 deficiency. We concluded that the characteristics of these mice are quite similar to those in human AMPD3 deficiency, including high ATP level in RBCs. Our results showed that AMPD3 deficiency does not rescue hemolytic anemia caused by decreased glycolytic ATP production, even though higher ATP levels were found in RBCs of A3(−/−) mice. In contrast, osmotic fragility was seen in AMPD3-deficient RBCs under a fasting condition. Additional studies are needed to characterize the physiological relevance of elevated ATP in AMPD3 deficiency and its physiological roles in tissues and cells other than RBCs, including those of the heart and brain.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Targeting construct and generation of AMPD3 knockout [A3(−/−)] mice

Genomic clones containing the AMPD3 gene were obtained from a mouse genomic library. The gene targeting strategy is illustrated in Fig. 1A. Briefly, the fragment containing exon 13 was replaced with a cassette containing a positively selectable marker, a neomycin resistance gene, under the control of the phosphoglycerate kinase promoter. For negative selection, a diptheria toxin gene was used.

Embryonic stem (ES) cells and derivatives were cultured without feeders in medium supplemented with FCS, 2-mercaptoethanol, and leukemia inhibitory factor. After transfection, colonies surviving positive/negative selection were isolated and screened by polymerase chain reaction (PCR) and Southern blot analysis to confirm that recombination had occurred on both sides of the gene. Chimeric mice were produced by microinjection of a targeted ES clone into C57BL/6 blastocysts and bred with female C57BL/6 mice. Mice with the targeted allele were backcrossed to C57BL/6 mice before analysis of homozygous mice. All animals had free access to food (CE-2, CLEAR) and water and were housed in a controlled SPF environment with a 12-h light–dark cycle and constant temperature (25 °C). The mice were then genotyped using PCR and Southern blotting analysis. All animal experiments were approved by the Committee on Animal Research of National Cerebral and Cardiovascular Center and were performed according to the guidelines for the protection of experimental animals of National Cerebral and Cardiovascular Center.

AMPD3 and pyruvate kinase double-deficient [(PKA (−/−, −/−)] mice

CBA-Pk-Islc/Pk-1slc [PK(−/−)] mice, a strain characterized by spontaneous development of PK deficiency along with decreased PK activity, anemia, splenomegaly, and increment of reticulocytes (Tsujino et al. 1998) were purchased from Japan SLC. Inc (Shizuoka, Japan). AMPD3 and pyruvate kinase double-deficient (PKA) mice were obtained by mating A3(−/−) with PK(−/−) mice. PK mouse genotyping was performed by PCR and Southern blotting analyses.

AMPD activity

Blood and tissues were homogenized with an extraction buffer (100 mm K-phosphate at pH 6.5, 180 mm KCL, 1 mm DTT). AMPD activity was determined using HPLC, as previously reported (Morisaki et al. 1992).

Measurement of nucleotide levels

Blood and fresh-frozen tissues were homogenized in 0.4 m perchloric acid. After centrifugation, the clear supernatant was neutralized, and 10 μL of neutralized supernatant was subjected to HPLC (Lachrom Elite, Hitachi, Japan) with a Capcell Pak C18 (Shiseido, Tokyo, Japan) column (Norman et al. 1994).

Reticulocyte staining

Blood was collected in heparinized tubes, and then 2.5 µL of 1% methylene blue was added to 2.5 µL of blood at 37 °C and allowed to stand for 15 min. The samples were subjected to a cytospin protocol before observation under a microscope.

Osmotic fragility test

Osmotic fragility was determined using the Parpart method. A 100-µL blood sample was added to a series of tubes containing 1 mL of saline solution (pH 7.0). The tubes were allowed to stand at room temperature for 30 min and then centrifuged to pellet the cells and absorbance of the supernatant was measured at 540 nm. The percentage of hemolysis was calculated by assuming hemolysis in water to be 100%.

Blood examinations

Blood glucose was measured using a blood glucose sensor (Kissei, Osaka, Japan). Determinations of Hb, RBC, WBC, HT, MCV, MCH, and MCHC levels were performed using standard methods.

Determination of phosphoribosylpyrophosphate synthetase (PRPPS) activity

Blood samples were collected in heparinized tubes, and PRPPS activity was determined using a previously reported method (Torres et al. 1994).

Statistical analysis

Statistical analysis was performed using the Student's two-tailed unpaired t-test, and the level of significance was set at P < 0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We express our thanks to Drs Hisaichi Fujii and Hitoshi Kanno at Tokyo Womens' Medical University for their useful suggestions. We also thank the members of the Department of Bioscience and Genetics for their technical support. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by grants from the Japan Science and Technology Corporation, the Ministry of Health, Labour and Welfare of Japan, and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO).

References

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
gtc12006-sup-0001-figureS1.docxWord document85KFigure S1 Osmotic fragility in PK deficient and PK/AMPD3 double-deficient RBCs.

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