Research Article: New insights on the regulation of the adenine nucleotide pool of erythrocytes in mouse models

Date Published: July 26, 2017

Publisher: Public Library of Science

Author(s): William G. O’Brien, Han Shawn Ling, Zhaoyang Zhao, Cheng Chi Lee, Tobias Eckle.


The observation that induced torpor in non-hibernating mammals could result from an increased AMP concentration in circulation led our investigation to reveal that the added AMP altered oxygen transport of erythrocytes. To further study the effect of AMP in regulation of erythrocyte function and systemic metabolism, we generated mouse models deficient in key erythrocyte enzymes in AMP metabolism. We have previously reported altered erythrocyte adenine nucleotide levels corresponding to altered oxygen saturation in mice deficient in both CD73 and AMPD3. Here we further investigate how these Ampd3-/-/Cd73-/- mice respond to the administered dose of AMP in comparison with the control models of single enzyme deficiency and wild type. We found that Ampd3-/-/Cd73-/- mice are more sensitive to AMP-induced hypometabolism than mice with a single enzyme deficiency, which are more sensitive than wild type. A dose-dependent rightward shift of erythrocyte p50 values in response to increasing amounts of extracellular AMP was observed. We provide further evidence for the direct uptake of AMP by erythrocytes that is insensitive to dipyridamole, a blocker for ENT1. The uptake of AMP by the erythrocytes remained linear at the highest concentration tested, 10mM. We also observed competitive inhibition of AMP uptake by ATP and ADP but not by the other nucleotides and metabolites tested. Importantly, our studies suggest that AMP uptake is associated with an erythrocyte ATP release that is partially sensitive to inhibition by TRO19622 and Ca++ ion. Taken together, our study suggests a novel mechanism by which erythrocytes recycle and maintain their adenine nucleotide pool through AMP uptake and ATP release.

Partial Text

The principal function of erythrocytes (red blood cells) is delivering oxygen to the body’s tissues, while simultaneously removing carbon dioxide from these tissues. These erythrocyte functions are in part modulated by adenine nucleotides. Both extracellular and intracellular levels of adenine nucleotides are tightly regulated by several well studied enzymes. Extracellularly, there is an ATPase (CD39) that converts ATP to ADP and then to AMP [1]. An extracellular ectonucleotidase (CD73) further dephosphorylates AMP to adenosine [2]. Adenosine is either taken up through nucleoside transporters such as ENT1/2 or quickly catabolized by adenosine deaminase (ADA). Intracellular adenosine can be phosphorylated by adenosine kinase, forming AMP. AMP can be further phosphorylated (with ATP as the phosphate donor) to produce ADP by adenylate kinase [3]. Another fate of intracellular AMP is deamination to inosine monophosphate (IMP) by AMP deaminase (AMPD). There are three tissue specific isoforms of AMPD; AMPD1, 2, and 3 are muscle, liver and erythrocyte-specific isoforms, respectively. In nucleated cells, IMP can be converted back to AMP by adenylosuccinate synthase and lyase. Thus, nucleotide degradation products can be salvaged to replenish the adenylate pool. Unlike nucleated cells, erythrocytes do not carry out de novo purine biosynthesis nor do they have a salvage pathway for IMP [4]. Erythrocyte AMP is mainly irreversibly catabolized by AMPD3, while a stable adenylate equilibrium is controlled by adenylate kinase. Having a similar regulatory environment for extracellular adenine nucleotides to other cell types, erythrocytes appear to have a unique mechanism to take up extracellular AMP efficiently [5][6].

Our work established a reliable procedure for AIHM, during which the core body temperature of the animal eventually drops to about 1°C above ambient temperature typically for 4–9 h and the animal appears to enter a state resembling that of suspended animation. Investigators who study natural hibernation and torpor of mammals have come to the conclusion that AIHM is an inducible form of torpor [24]. At the gene expression level, we found that AIHM involves changes in the expressions of a relatively small number of genes. These changes are largely restored within 48 h post-induction of AIHM, providing molecular evidence that AIHM is a safe and reversible process. Interestingly, the circadian clock was found to be largely stalled at the gene expression level during AIHM, a feature also observed in natural hibernations [25]. Our metabolomics studies revealed that the 5’-AMP administered was largely catabolized by the time the animals entered AIHM and their urea cycle appeared to be functional, helping to avoid ammonia toxicity [26]. For potential applications of AIHM, one of our studies demonstrated that AIHM induces a reversible deep hypothermia that reduces ischemia/reperfusion damage following myocardial infarct [9]. Another study demonstrated that whole body cooling increased stabilization of a temperature-sensitive Cystic Fibrosis (CF) mutant protein, ΔF508-CFTR, improved its functions, alleviated CF pathological phenotypes and decreased mortality in CF mice [27]. Similar AIHM procedures are now used by independent research groups to cool experimental animals of various disease models [28][29][30][31][32].




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