Increased Anion Exchanger-1 (Band 3) on the Red Blood Cell Membrane Accelerates Scavenging of Nitric Oxide Metabolites and Predisposes Hypertension Risks

Abstract

The erythrocyte membrane is highly specialized with ∼1 million anion exchanger-1 (AE1) per cell for rapid membrane permeation of HCO3-(aq), as most blood CO2(g) is carried in this hydrated anionic form. People with the GP.Mur blood type have more AE1 on their erythrocyte membrane, and they excrete CO2(g) more efficiently. Unexpectedly, GP.Mur/increased AE1 is also associated with higher blood pressure (BP). To solve this, we knocked the human GYP.Mur gene into C57BL/6J mice at 3'-UTR of GYPA to generate GPMur knock-in (KI) mice. KI of human GYP.Mur increased murine AE1 expression on the red blood cells (RBC). GPMur KI mice were naturally hypertensive, with normal kidney functions and lipid profiles. Blood NO3- [the stable nitric oxide (NO) reservoir] was significantly lower in the GPMur mice. GPMur KI also accelerated AE1-mediated NO2- influx into the RBCs and intraerythrocytic NO2-/NO processing. From tests with different categories of antihypertensives, hypertension in GPMur mice responded best to direct arterial vasodilator hydralazine, suggesting that vasodilator deficiency is the leading cause of "GPMur/AE1-triggered hypertension." In conclusion, we showed that GPMur/increased AE1 predisposed hypertension risks. Mechanistically, higher AE1 expression increased RBC membrane permeability for NO2- and consequently accelerated erythroid NO2-/NO metabolism; this is associated with lower NO bioavailability and higher BP. As hypertension affects a quarter of the world population and GP.Mur is a common Southeast Asian (SEA) blood type, this work may serve as a primer for "GPMur (biomarker)-based" therapeutic development for hypertension.

Keywords: GP.Mur (Mi.III); Miltenberger blood type; anion exchanger-1 (AE1; anion transport; band 3); blood pressure; glycophorin; hypertension; nitric oxide; nitrite; red blood cell.

Graphical Abstract

Figure 1.

Design of the human GPMur knock-in mouse (GPMur KI) model by CRISPR/Cas9-mediated DNA cleavage and homologous recombination with a replacing ssDNA on the mouse Gypa allele. The cleavage site for the designed CRISPR/Cas9-mediated recombination was located after the stop codon of mouse Gypa. The target regions of the primer pairs for PCR genotyping are indicated. Abbreviations: 3′HR, 3′ homologous arm; 5′HR, 5′ homologous arm; IRES, polio internal ribosome entry site.

Figure 2.

The expression of human GPMur in the KI mice was verified by (A) RT-PCR, (B) flow cytometry, and (C) confocal imaging. (A) RT-PCR revealed the presence of human GYP.Mur transcript in the peripheral blood of GPMur KI mice, and not that of the control mice (top). The housekeeping gene GAPDH served as the experimental (RT-PCR) control (bottom). (B) GPMur flow cytometry using anti-GPMur Ab (clone PJ90929) revealed the presence of GPMur protein in the RBCs from GPMur KI mice, compared to age-matched control mice. (C) TER-119-stained murine erythrocytes were visualized by confocal microscopy. Scale bar, 10 μm.

Figure 3.

Human GPMur enhanced murine AE1 expression on the RBC membrane. (A) The immunoblot examined AE1 expression levels in the RBC membrane lysates from 6 GPMur KI and 6 control mice, and found more monomeric and dimeric AE1 in the GPMur than the control mice. The circle symbols indicate monomers and dimers of AE1. (B) Mouse AE1 ELISA verified more AE1 present in the erythroid membrane fraction of the GPMur KI than the control mice. Each dot represents the measurement of a mouse. Shown mean ± SD. **P < 0.01 deemed significant by an unpaired t-test.

Figure 4.

GPMur/more AE1 lowered systemic NO₃⁻ reservoir. The levels of blood plasma nitrate and nitrite from GPMur KI vs. age-matched control mice were measured. Each dot represents the datum of a mouse measurement. The horizontal bars indicate mean ± SD. *P < 0.05 deemed statistical significance.

Figure 5.

Intracellular DAF-FM labeling of nascent NO production from NO₂⁻ revealed the influence of GPMur/anion transporter in erythroid NO₂⁻/NO processing. (A) The experiment rationale: To test whether higher AE1 expression (as in GPMur KI) could influence erythroid NO₂⁻/NO processing, RBCs were first loaded with NO-sensitive DAF-FM probe, which fluoresces upon binding to NO. Nitrite passes through AE1 to enter RBCs, where it can be converted to NO by deoxyHb. Though nascent NO synthesized from intraerythrocytic nitrite reduction should be scavenged by oxyHb at an extremely fast rate, a fraction of nascent NO is trapped upon binding to DAF-FM, which allows assessment of erythroid NO₂⁻/NO processing. (B) Top panels show the snapshots of DAF-FM-loaded RBCs from GPMur KI and the control mice right before the addition of nitrite, and at the 6th, 10th, and 20th minute following the nitrite treatment. Left: the fluorescence intensities of 20 DAF-FM-loaded RBCs from each mouse were averaged and plotted in mean ± SEM at each time point. Right: DAF-FM fluorescence intensities were normalized to maximal 100%.

Figure 6.

GPMur/more AE1 accelerated and increased erythroid NO₂⁻/NO metabolism. (A) Before normalization of the DAF-FM fluorescence intensities, RBCs from the GPMur KI mice (n = 6) had significantly more nascent NO generated from nitrite reduction, compared to RBCs from the control mice (n = 6). (B) After normalization of the DAF-FM intensities, the rates of NO produced from nitrite reduction in the RBCs of the control mice (n = 6) were significantly slower, compared to the rate in the RBCs of the GPMur KI mice (n = 6). The horizontal bars indicate mean ± SD. **P < 0.01 and *P < 0.05 deemed statistical significance.

Figure 7.

Antihypertensive drug tests revealed GPMur KI as a novel hypertension murine model. (A) Among the popular antihypertensives tested, hydralazine and amlodipine were more effective in BP reduction for GPMur KI (labeled gray) than the age-matched, control mice (labeled black); in contrast, valsartan exerted similar BP-reducing effects. BP at t = 0 was measured right before oral gavage of the drug. For each drug tested, the effects of GPMur/AE1 on BP reduction within 10 h post-treatment were assessed by ANCOVA (*P < 0.05 and **P < 0.01 deemed statistically significant). The number of mice per group was indicated in parentheses inside the legend box. Shown mean ± SEM. (B) The dose responses of hydralazine were shown as SBP changes in 1-h hydralazine treatment (∆SBP). Fitting of the dose response curve of hydralazine converged for GPMur KI, but failed for the control mice (shown as a dotted line).

Figure 8.

The dual roles of erythroid anion transport in NO and BP regulation and in CO₂ respiration. (Top) From the comparative studies using the GPMur KI mice, the driving force for NO₂⁻ influx/scavenging is stronger with a more anion-permeable erythroid membrane. NO₂⁻_(aq) can be converted to NO_(g) and other forms of NO metabolites inside the RBCs. Nitric oxide gas inside erythrocytes is extremely rapidly converted to stable NO₃⁻_(aq) by oxyHb. NO₃⁻_(aq) can permeate in and out of the RBC through AE1. Lower NO₃⁻_(aq) is observed in the bloodstream of GPMur KI mice. Reactive NO₂⁻ that is constantly converted to even more reactive vasodilating NO and other NO metabolites may be supplied from NO₃⁻ (the main storage form of NO_(g) in the body) by nitrate reductase in the microbiome (dotted arrows). GPMur/more AE1 RBCs scavenges NO₂⁻ at faster rates and uses up NO₃⁻ more rapidly, which may trigger higher BP. In the illustration, AE1 is symbolized as the gates or blocks on the RBC membrane; the blood NO₃⁻ reservoir is symbolized in dotted boxes. (Bottom) AE1 on the RBC membrane transports HCO₃⁻_(aq) bidirectionally, depending on the bicarbonate gradients across the cell membrane (which differ in different locations in the body). As RBCs circulate to the lung capillaries, where CO_2(g) pressure drops, blood HCO₃⁻_(aq) rushes into the RBCs through AE1 to be converted to CO_2(g) for expiration by intraerythrocytic carbonic anhydrase II (CAII). The CO₂ gas is not charged and can thus diffuse and leave the RBCs through lipid bilayer and transporters/channels. More AE1 on the RBC membrane (GPMur) accelerates influx of HCO₃⁻_(aq) and conversion to CO_2(g) as RBCs circulate to the pulmonary capillaries. Note that the rate of erythroid anion transport via AE1 is much slower than the rates of the reactions facilitated by these intracellular enzymes, i.e. deoxyHb (top) and CAII (bottom), and thus AE1-mediated anion flux is the rate-limiting step in both physiologic phenomena.