The environmental factors are constantly fluctuating. Therefore, in order for the trees to survive, they have to use their own pathway in acclimating to the ups and downs. Humans have to also apply their systemic skills to adapt to the changes during low oxygen levels or they can always accept the hypoxic danger. The oxygen delivery to the tissues during hypoxia is necessary for the survival of the species. The answer to this tactical way of acclimation during environmental instability is by depending on the adenosine signaling pathway. A study by Song et al., explains the evolution behind the decreased acute mountain sickness (AMS). Song et al. found out that within erythrocytes, a forward signaling network that helps in adapting to initial hypoxia and retention upon re-exposure1.
A lot of research has been going on for a century to understand the adaptive pathway in response to high altitude. Here, the study provides both human and mouse experimental evidence on how the body responses to hypoxia. After an exposure to hypoxia, noticeable changes were documented. There was an increase in the levels of sCD73 (soluble ecto-5′-nucleotidase) and ATP and reduced levels of equilibrative nucleoside transporters (ENTs). The erythrocyte equilibrative nucleoside transporter 1 (eENT1) is one of the proteins that has a role in extracellular adenosine uptake. Another study was done by Horenstein et al., provides additional insights on CD73 and ATP working together to form adenosine2. Thus, the two factors in addition to adenosine A2B receptor (AdoRA2B) allow the purinergic signaling network to be activated. The activation of the network encourages PKA phosphorylation, ubiquitination and degradation of eENT1.
At first, the study was focused on investigating the levels of plasma adenosine and sCD73 upon initial exposure and re-exposure. They found that the purinergic components in the plasma from the human volunteers higher upon re-ascent compared with the first hypoxic exposure (ALT1). Next, they performed a Pearson Correlation analysis to find if there was a decrease in the AMS-C composite score between the initial hypoxic exposure and re-exposure. Upon re-ascent, there was an increase in the plasma adenosine levels which is found to be correlated to the adaptation process.
Even though adenosine is a powerful hypoxic-signaling radar, the case is destroyed under normoxia. It is attacked and transported into the cells by ENTs. ENTs are important in regulating the levels of adenosine and to investigate the process in vivo, they injected mice with C14 adenosine. Within a minute, the adenosine was transported inside erythrocytes. Erythrocytes, took in 90% of the injected adenosine. After finding which cells, it is time to find the transporter. They used nitrobenzylmercaptopurine, a specific inhibitor of ENT1 and dipyridamole, a general ENT inhibitor, in comparison with untreated cells. There was no uptake of adenosine within the global ENT1-deficient mice (Ent1−/−) compared to (Ent2−/−). Next, they generated erythrocyte-specific ENT1 and ENT2 knockouts to further investigate which protein was absent. Western blot analysis showed both ENT1 and ENT2 were absent which means they have been deleted. Upon deletion of the eENT1 from the Ent1flox/flox/EpoRCre mice and exposing them to acute hypoxia for about 72 hours, adenosine levels were found to rise very high compared to the controlled cells (EpoRCre). So, the removal of eENT1 did cause an accumulation of extracellular adenosine under acute hypoxia. Thus, reducing tissue damage and inflammation which could have been caused by the hypoxic exposure.
Another investigation was done with ENT1 knockouts for 72 hours under hypoxic exposure. There was an increase in the Plasma ATP and sCD73 levels. Under normoxia, the two factors were lower compared to hypoxia. In addition to degraded eENT1, they work together to rise the plasma adenosine levels. They performed immunoprecipitation (IP) using an antibody specific for ubiquitin followed by a western blot analysis against ENT1. Upon hypoxia exposure, they found that polyubiquitinated ENT1 ((Ub) n-ENT1)) levels in lysates were elevated for two days and then degraded on the third day. In previous studies, AdoRA2B and PKA are reported to be involved in ubiquitination, so they had to dig a little deeper. They found that phosphorylated PKA is required for AdoAR2B-induced ubiquitination, followed by a proteasome degradation of ENT1. Next they treated wild type mouse erythrocytes with AdoRA2B agonist (Bay60-6583). Both the Bay60-6583 and forskolin treatments induced ubiquitination, translocation and then degradation of ENT1. A similar test was done on the erythrocytes isolated from the human volunteers during AltitudeOmics study. There was an increase in PKA phosphorylated ENT1 and ubiquitination upon exposure to hypoxia.
For further studies, they traced protein levels of ENT1 by labelling cells with N-hydroxysuccinimide (NHS) biotin before the first exposure to hypoxia. Upon first hypoxic exposure, the co-immunoflurosence staining showed a decreased in that membrane-anchored eENT1. This also supports the previous facts of lower ENT1, followed by higher levels of plasma adenosine. Although ENT1 protein in human erythrocytes is reduced by initial hypoxia and further reduced by re-exposure, the case might be different for the mouse erythrocyte. The life span of a mouse erythrocyte is 55 days. The low eENT1 protein levels in past cells will be replaced with new born cells. The levels are normal as if no hypoxia has occurred.
As previous studies lacked evidence, this study provides evidence from both the human volunteers and the mice in order to produce the current valuable data. The humans in general are surrounded by challenges and hypoxia is one of them. If purines didn’t exist, then the purinergic memory would never existed in this study. The purines have their own special receptor system such as adenosine A2B receptor (AdoRA2B) which is part of the purinergic signaling network. Now, understanding the purinergic signaling network that is behind the adaptation process, will enable us to find therapeutics to treat exposures to hypoxia or even treat it in advance. The environment is unstable and the world may face future difficulties related to hypoxia such as limited oxygen supplies or rising water levels causing people to move to high mountains, etc.
- Song, A. et al. Erythrocytes retain hypoxic adenosine response for faster acclimatization upon re-ascent. Nature Communications 8, 14108 (2017).
- Horenstein, A. L. et al. A CD38/CD203a/CD73 ectoenzymatic pathway independent of CD39 drives a novel adenosinergic loop in human T lymphocytes. Oncoimmunology 2, (2013).
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