A role of erythrocytes in adenosine monophosphate initiation of hypometabolism in mammals

Abstract

Biochemical and mechanistic aspects into how various hypometabolic states are initiated in mammals are poorly understood. Here, we show how a state of hypometabolism is initiated by 5'-AMP uptake by erythrocytes. Wild type, ecto-5'-nucleotidase-deficient, and adenosine receptor-deficient mice undergo 5'-AMP-induced hypometabolism in a similar fashion. Injection of 5'-AMP leads to two distinct declining phases of oxygen consumption (VO(2)). The phase I response displays a rapid and steep decline in VO(2) that is independent of body temperature (T(b)) and ambient temperature (T(a)). It is followed by a phase II decline that is linked to T(b) and moderated by T(a). Altering the dosages of 5'-AMP from 0.25- to 2-fold does not change the phase I response. For mice, a T(a) of 15 degrees C is effective for induction of DH with the appropriate dose of 5'-AMP. Erythrocyte uptake of 5'-AMP leads to utilization of ATP to synthesize ADP. This is accompanied by increased glucose but decreased lactate levels, suggesting that glycolysis has slowed. Reduction in glycolysis is known to stimulate erythrocytes to increase intracellular levels of 2,3-bisphosphoglycerate, a potent allosteric inhibitor of hemoglobin's affinity for oxygen. Our studies showed that both 2,3-bisphosphoglycerate and deoxyhemoglobin levels rose following 5'-AMP administration and is in parallel with the phase I decline in VO(2). In summary, our investigations reveal that 5'-AMP mediated hypometabolism is probably triggered by reduced oxygen transport by erythrocytes initiated by uptake of 5'-AMP.

FIGURE 1.

Relationship between VO₂, T_b, and T_a following 5′-AMP injection. a and b, simultaneous measurement of T_b and VO₂ of mice given saline or 5′-AMP (0.5 mg/g, indicated by arrow) in individual metabolic chambers at T_a of 23 and 15 °C. Each mouse used in the study is represented by the same color trace for VO₂ and T_b. Sampling time for both T_b and VO₂ was about 8 min. Note that the phase I (PI) decline of VO₂ is steep and rapid after administration of 5′-AMP. In contrast, the phase II (PII) VO₂ decline is gradual and coincides with declining T_b. The colors reflect VO₂ and T_b of independent measurement of different animals given either saline or 5′-AMP. c, respiration rate of mice (n = 4) with T_b ∼16 °C compared with 37 °C. d, heart rate of mice (n = 3) with T_b 16 °C compared with 37 °C. *, p < 0.001, paired t test. Error bars, S.E.M.

FIGURE 2.

DH as a function of 5′-AMP dosage. a–d, simultaneous measurement of T_b and VO₂ of mice given different dosages of 5′-AMP and maintained at T_a of 15 °C. The different colors in the graph reflect VO₂ and T_b of different animals used in the study. e, comparison of VO₂ levels in mice given a similar amount (0.5 mg/g) of either 5′-AMP, 5′-CMP, or 5′-GMP. f, arousal from DH and the rise in T_b of mice (n = 4) maintained at various T_a values.

FIGURE 3.

Loss of adenosine receptors or ecto-5′-nucleotidase deficiency does not block DH by 5′-AMP. a, the VO₂ profiles of ectonucleotidase/CD73-deficient (n = 2) and wild type mice following 5′-AMP (0.5 mg/g) administration. b, individual T_b of ectonucleotidase/CD73-deficient and wild type mice (n = 12) after 1 h at 4 °C T_a with injection of 0.125 mg/g 5′-AMP. c, top, T_b time course of mice (n = 4) with the adenosine receptor genotype A₁^−/−, A_2a^−/−, A_2b^−/−, or A₃^−/− or wild type were given 0.5 mg/g 5′-AMP after 1 h at 4 °C T_a. Bottom, T_b of mice of the same genotype given saline after 1 h at 4 °C T_a. Error bars, S.E.M.

FIGURE 4.

Increased production of 2,3-bisphosphoglycerate induced by 5′-AMP uptake by erythrocytes. a, levels of 2,3-bisphosphoglycerate in erythrocytes/red blood cells (RBC) obtained from mice after administration of 5′-AMP (*, data shown as mean ± S.E.M. (error bars), p < 0.05, n = 6). b and c, glucose and lactate levels in erythrocytes isolated from 5′-AMP and untreated mice (data shown as mean ± S.E.M. p < 0.01, n = 4).

FIGURE 5.

Uptake of 5′-AMP by erythrocytes and increased deoxyhemoglobin levels. a, uptake of radiolabeled [¹⁴C]adenosine or 5′-[¹⁴C]AMP by isolated erythrocytes in vitro. Autoradiograph showing the TLC analysis of erythrocyte extracts compared with known radiolabeled purine standards. Shown are representative HPLC chromatograms of purine products extracted from blood obtained from mice in euthermia (b), in spontaneous arousal (c), and during DH (d) and an animal in DH given a second injection of 0.5 mg/g 5′-AMP and sacrificed 2 h later (e). HPLC of purine standards was used to determine the identity of the various peaks. f, absorbance spectrum of whole blood from mice (n = 2) treated with 5′-AMP (dashed lines) or untreated (solid lines). Blood was taken 10 min after intraperitoneal administration of 5′-AMP (0.5 mg/g).

FIGURE 6.

A proposed model for 5′-AMP-mediated hypometabolism via erythrocytes. The model indicates altered levels of glycolytic intermediates and adenylates observed. In the erythrocytes, uptake of 5′-AMP drives the production of ADP at the expense of ATP. In turn, glycolytic steps dependent on ATP at hexokinase (HK) were slowed, resulting in the buildup of glucose and decreased production of lactate. The reduction in glycolytic rate will drive the production of 2,3-BPG from 1,3-BPG by the enzyme bisphophoglycerate mutase to stabilize intracellular ATP levels. The increase in 2,3-BPG allosterically reduces the affinity of hemoglobin (Hb) for oxygen, which could explain the rapid and steep decline in VO₂ illustrated by phase I and increased deoxyhemoglobin levels.