Erythrocytes retain hypoxic adenosine response for faster acclimatization upon re-ascent

Abstract

Faster acclimatization to high altitude upon re-ascent is seen in humans; however, the molecular basis for this enhanced adaptive response is unknown. We report that in healthy lowlanders, plasma adenosine levels are rapidly induced by initial ascent to high altitude and achieved even higher levels upon re-ascent, a feature that is positively associated with quicker acclimatization. Erythrocyte equilibrative nucleoside transporter 1 (eENT1) levels are reduced in humans at high altitude and in mice under hypoxia. eENT1 deletion allows rapid accumulation of plasma adenosine to counteract hypoxic tissue damage in mice. Adenosine signalling via erythrocyte ADORA2B induces PKA phosphorylation, ubiquitination and proteasomal degradation of eENT1. Reduced eENT1 resulting from initial hypoxia is maintained upon re-ascent in humans or re-exposure to hypoxia in mice and accounts for erythrocyte hypoxic memory and faster acclimatization. Our findings suggest that targeting identified purinergic-signalling network would enhance the hypoxia adenosine response to counteract hypoxia-induced maladaptation.

Figure 1. Elevated circulating purinergic components are associated with acclimatization and subsequent hypoxic adenosine response upon re-ascent.

(a) Schematic illustration of Human AltitudeOmics Study. (b) Plasma adenosine levels of samples collected at different time points (n=21, error bar: mean±s.e.m., *P<0.05, t-test). (c) sCD73 activities of plasma samples collected at different time points (n=21, error bar: mean±s.e.m., *P<0.05, **P<0.001, t-test). (d) Changes of plasma adenosine was significantly correlated with improved acclimatization judged by negative correlation with changes of AMS-C composite scores (acute mountain sickness; n=21, Pearson correlation r=−0.64, P=0.003). (e,f) Plasma adenosine is significantly associated with improved acclimatization at Post7/21 in comparison with ALT1 (e) associated with LLQ-Headache (ordered logistic regression, n=21, P<2.2e−16, FDR<2.2e−16), (f) associated with LLQ-Dizzy (ordered logistic regression, n=21, P<2.2e−16, FDR<2.2e−16). FDR=false discovery rate.

Figure 2. eENT1 is the major factor eliminating extracellular adenosine.

(a) In vivo adenosine uptake assay, comparison is between plasma and blood, n=4, P<0.05. (b) In vitro adenosine uptake assay, *Comparison between RBC from ENT1^−/− and WT, n≥4, P<0.001; #comparison between dipyridamole or NBMPR and DMSO, n=4, P<0.001. (c) In vitro adenosine uptake assay using human erythrocytes, n=4, *P<0.05. (d) Plasma adenosine from hypoxia-treated Ent1^flox/flox/EpoRCre or EpoRCre mice. *: between normoxia and 48-h hypoxia, n=4, P<0.05. **between 24- and 72-h hypoxia, n=4, P<0.05. ⁺between normoxia and 24-, 48- or 72-h hypoxia, n=4, P<0.05. ⁺⁺between EpoRCre and Ent1^flox/flox/EpoRCre after 48- or 72-h hypoxia, n=4, P<0.05. (e) Reduced tissue hypoxia was observed in Ent1^flox/flox/EpoRCre compared with EpoRCre after 72-h hypoxia treatment by HypoxiaProbe, red: HypoxiaProbe, blue: DAPI; scale bar, 200 μm. (f–h) Deletion of eENT1 protects from acute lung injury indicated by (f) total cell number (n=4, *P<0.05), (g) albumin (n=4, *P<0.05) and (h) IL-6, (n=4, *P<0.05) in BALF from 72-h hypoxia-treated animals. (i) Pulmonary neutrophil accumulation by MPO assay in lung tissues from 72-h hypoxia-treated animals (n≥4, *P<0.05). (j) H&E-stained lung section from 72-h hypoxia-treated animals (edema, red arrows); scale bar, 500 μm. All error bars: mean±s.d. and t-test.

Figure 3. Hypoxia downregulates eENT1 through adenosine–ADORA2B–ubiquitin signalling.

(a) Hypoxia decreased eENT1 activity in erythrocytes from hypoxia-treated animals as judged by in vitro adenosine uptake assay, n≥4, *comparison between normoxia and 48-h hypoxia-treated animals, P<0.05; **comparison between normoxia and 72-h hypoxia-treated animals, P<0.001, error bar: mean±s.e.m. (b) sCD73 activity in Ent1^flox/flox/EpoRCre or EpoRCre mice with or without hypoxia treatment, n=4, error bar: mean±s.d., P<0.001, t-test. (c) Plasma ATP in Ent1^flox/flox/EpoRCre or EpoRCre mice with or without hypoxia treatment, n=4, error bar: mean±s.e.m., P<0.05. (d) Erythrocyte ENT1 is reduced during hypoxia treatment through ubiquitination by western blot against ENT1 (left panel, β-actin was used as loading control, n=3, error bar: mean±s.d., ⁺P<0.05). Right panel, IP ubiquitin followed by western blot against ENT1, *non-specific bands. (e) Hypoxia-mediated downregulation of eENT1 activity (left panel) and eENT1 protein (immunofluorescence against ENT1, red colour: ENT1, right panel; scale bar, 5 μm) were observed in C57BL/6 mice, but not CD73^−/− or AODRA2B^−/− mice when we treated these animals under hypoxia (8% oxygen) for 72 h, n≥6, error bar: mean±s.d., P<0.05). (f) Adenosine signalling through ADORA2B downregulates eENT1 activity as judged by in vitro adenosine uptake assay (n=3, error bar: mean±s.d., *P<0.05). t-test is used for all comparisons in this figure.

Figure 4. ADORA2B–PKA–ubiquitin–proteasomal degradation of eENT1 in mice.

(a) IP–western blot using phospho-PKA subtract antibody pull-down proteins from RBC lysate followed by western blot against ENT1, β-actin as an input control, n=3, *compared with normoxia, P<0.05, t-test. (b,c) In vitro adenosine uptake assay. Activation of ADORA2B by Bay 60-6583 (B60) or activation of PKA by forskolin decreases eENT1 activity that can be blocked by H-89, MG132 or BT (Bortezomib). (b) By B60, n=3, *comparison between DMSO and B60 treatment, P<0.05; ⁺ and ⁺⁺comparison between B60 and B60 plus inhibitor, ⁺P<0.05, ⁺⁺P<0.001. (c) By forskolin, n=3, *comparison between DMSO and forskolin treatment, P<0.05; ⁺ and ⁺⁺comparison between forskolin and forskolin plus inhibitor, ⁺P<0.05, ⁺⁺P<0.001. (d) Immunofluorescence, activation of ADORA2B or PKA leads to eENT1 ubiquitination and degradation. B60 and forskolin induced protein ubiquitination in a time-dependent manner and the effect of B60 can be blocked by H-89 (middle panels). B60 and forskolin induced eENT1 translocation to the cytosol after 3-h treatment and degradation after 6-h treatment. H-89 blocked B60-induced translocation and degradation of eENT1. BT blocked B60- and forskolin-induced degradation of eENT1, but not the translocation (upper panels). Red: eENT1, green: ubiquitin, scale bar: 2 μm. Error bars: mean±s.d., t-test.

Figure 5. PKA phosphorylation-mediated ubiquitination and porteasomal degradation of eENT1 in humans at high altitude and upon re-ascent.

(a) IP phospho-PKA substrate followed by western blot against ENT1, (IP–western blot is representative image from three experiments using three sets of RBC samples from the Human AltitudeOmics Study), P-ENT1: PKA phosphorylated ENT1. (b) IP ubiquitin followed by western blot against ENT1, n=3, (IP–western blot was performed using RBC samples from three subjects), *: compared with SL, error bars: mean±s.d., P<0.05, t-test. (c) Hypoxia-induced downregulation of eENT1 protein levels in erythrocyte samples judged by western blot against ENT1, n=3 (RBC samples from three subjects), *compared with SL, error bars: mean±s.d., P<0.05, t-test.

Figure 6. Erythrocyte has hypoxic adenosine response through hypoxia-mediated downregulation of eENT1.

(a) Experimental illustration for acclimatization hypoxic adenosine response. (b) Erythrocyte life span was indicated by biotin NHS labelling and revealed by flow cytometry. (c) eENT1 changes during hypoxia treatment as judged by immunofluorescence, red: ENT1, green: Biotin; scale bar, 5 μm. (d) eENT1 activity (n=4, *P<0.05). (e) Plasma adenosine levels. n=4 for all groups, *between normoxia and first hypoxia, P<0.05; ⁺between Post3 normoxia and Post3 re-hypoxia, P<0.05; ⁺⁺between Post50 normoxia and Post50 re-hypoxia, P<0.05; **between first hypoxia and Post3 re-hypoxia, P<0.05. Error bars: mean±s.d., t-test.

Figure 7. Working model.

Our findings support a working model that elevated sCD73-mediated increased adenosine signalling via ADORA2B results in PKA-mediated phosphorylation, ubiquitination and proteasomal degradation of ENT1 in the erythrocytes of humans at high altitude and mice exposed to hypoxia. Thus, purinergic-signalling components play an important role in enhancing accumulation of plasma adenosine and thus promote initial adaptation to hypoxia in a feed-forward manner. Moreover, these newly identified erythrocyte purinergic components retain hypoxic adenosine response to promote quicker and higher elevation of plasma adenosine and thereby allow for rapid acclimatization upon re-ascent. Our findings offer multiple innovative therapies to counteract hypoxic challenge under physiological and pathological conditions.