Research ArticleCELLULAR ENERGY

Accelerating metabolism and transmembrane cation flux by distorting red blood cells

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Science Advances  18 Oct 2017:
Vol. 3, no. 10, eaao1016
DOI: 10.1126/sciadv.aao1016
  • Fig. 1 13C NMR (100.61 MHz) spectral time course of human RBCs metabolizing [1,6-13C]d-glucose in relaxed gelatin gel.

    Suspension medium was 60 mM NaOH, 110 mM NaCl, 10 mM KCl, and 10 mM CaCl2 at pH 7.4 and 20°C with 9.95 mM [1,6-13C]d-glucose. Resonance assignments were as follows: 91.3 and 95.1 ppm, C1 of α and β anomers of [1,6-13C]d-glucose; 65.6 ppm, C3 of 23BPG; 59.8 ppm, partially resolved C6 of α and β anomers of [1,6-13C]d-glucose; 19.3 ppm, methyl carbon of [3-13C]l-lactate; 13.1 ppm, methyl carbon of [6-13C]l-methionine added as an intensity reference; and 0 ppm, natural-abundance 13C in a silicone rubber tube used as the chemical-shift reference. A ~30° excitation pulse was used, with a recovery delay of 2 s per free induction decay (FID), to acquire 816 transients per spectrum, giving a total acquisition time of 30 min. For clarity, only every 10th spectrum is shown.

  • Fig. 2 Reversible effect of mechanical deformation of RBCs on their glycolytic rate.

    [3-13C]l-Lactate production by RBCs in gelatin gel at 20°C was recorded from 9.95 mM [1,6-13C]d-glucose in the same suspension medium, as for Fig. 1. As shown by the insets, the RBC/sample was initially relaxed (red circles), then 70% compressed (green circles), and relaxed again (blue circles). The amount of produced [3-13C]l-lactate was calculated from the peak integrals of its methyl resonance, relative to that of standard [13CH3]l-methionine, in the sequential 13C NMR spectra. For each spectrum, 812 transients were acquired for a total time of 30 min using a ~30° excitation pulse with a duration of 17.5 μs and an intertransient delay of 2 s. Before Fourier transformation, a decaying exponential window function with a line-broadening factor of 5 Hz was applied. The data were imported into a program written in Mathematica (49) that automatically performed spectral peak fitting, thus allowing us to calculate peak integrals. Calculation of the [3-13C]l-lactate concentration was performed by calibration of the peak integrals relative to the standard compound ([13CH3]l-methionine present at a concentration of 5.32 mM). The solid lines are least-squares regression fits to the data, and the dashed lines are extrapolations that emphasize the major change in slope that reversibly occurred on transitioning between the stages (see text). Two-tailed Student’s t tests indicated no statistically significant difference at P < 0.05 for the first and second relaxed-state slopes, and significant difference at P < 0.01 with respect to the middle slope.

  • Fig. 3 Reversible effect of compression on efflux of Cs+ from human RBCs.

    Cs+-loaded RBCs were suspended in gelatin gel that was relaxed (red circles), 70% compressed (green circles), and then relaxed again (orange circles). Net 133Cs+ efflux was measured with 133Cs NMR (52.48 MHz) spectroscopy at 20°C by recording 12 sequential spectra in each stage. The sample was from the same batch of RBCs used for fig. S2 and treated in the same manner. Fitted slopes obtained with NonlinearModelFit in Mathematica, for the respective stages, were (1.7 ± 0.5) × 10−4 min−1 (9.1 ± 0.9) × 10−4 min−1 and (0.53 ± 0.04) × 10−4 min−1. With an initial intracellular [Cs+] of 16.5 mM, these rates corresponded to respective effluxes of 2.8 ± 0.8 μmol (liter RBC)−1 min−1, 15.0 ± 1.5 μmol (liter RBC)−1 min−1, and 0.80 ± 0.07 μmol (liter RBC)−1 min−1 (where “±” denotes SE, and the second-stage rate is significantly different from the first at P < 0.05). The two inset spectra correspond to the third point in the respective time course, where the dots are the data points and the solid line is a least-squares fit of a line-shape function (described in Materials and Methods) to the spectra, performed with NonlinearModelFit in Mathematica. Note that the chemical shift scale runs from left to right according to the mathematical convention used in Mathematica, but this is opposite to the convention in NMR spectroscopy. The apparent “jumps” in flux that arose at the beginning and end of the compression stage were an artifact of the broad baseline in the compressed gel (see the Supplementary Materials under “133Cs+ efflux from RBCs in gels” for further comment).

  • Fig. 4 Stimulation of glycolysis by yoda1.

    13C NMR (100.61 MHz) spectra of human RBCs metabolizing [1,6-13C]d-glucose in a gel-free suspension at 37°C and hematocrit (Ht) = 75%, (A) without and (B) with yoda1. For each spectrum, 176 transients of 8192 data points were acquired in 6.87 min using a ~30° excitation pulse of 17 μs and an intertransient delay of 2 s. Before Fourier transformation, a decaying exponential window function with a line-broadening factor of 8 Hz was applied. The RBC suspension medium was as follows: 30 mM CsCl, 10 mM KCl, and 114 mM NaCl, with an osmolality of 282 mosmol kg−1; 8.2 mmol (liter sample)−1 or 10.39 mmol (liter aqueous space)−1 [1,6-13C]d-glucose; 5.13 mmol (liter sample)−1 or 4.05 mmol (liter aqueous space)−1 [6-13C]l-methionine; 2.1 μl of 1 M stock, giving 0.88 mmol (liter aqueous space)−1 or 2.8 mmol (liter extracellular aqueous space)−1 [Ca2+]; and 2.0 μl of 28.15 mM stock in DMSO, giving 19 μmol (liter sample)−1 yoda1. Because the mean RBC volume was 86 fl, there were 2.61 × 1010 cells in the sample, implying 4.4 × 108 yoda1 molecules per cell.

  • Fig. 5 Schematic representation of the Ca2+-mediated linkage between activation of Piezo1 by mechanical distortion of the RBC and stimulation of its glycolysis.

    The various participants in this complex response are as follows: GLUT1, which is the glucose transporter required to deliver this fuel molecule to the cytoplasm; Ca-ATPase, which responds to an increase in [Ca2+] by catalyzing the hydrolysis of one molecule of ATP per Ca2+ ion ejected from the cytoplasm; and Na,K-ATPase, which constitutively pumps three Na+ ions from the cell while simultaneously importing two K+ ions with the concomitant hydrolysis of one molecule of ATP; in the physiological operation of the RBC, this reaction consumes ~40% of the free energy derived from glycolysis (28, 32). Capnophorin (also called Band 3) catalyzes the one-for-one exchange of the anions, Cl and HCO3, and is central to the attainment of bulk electroneutrality. The monocarboxylate transporter (MCT) mediates the facilitated diffusion of lactate across the RBC membrane. Its operation was evident from the splitting of the [3-13C]l-lactate resonance that indicated two compartments (inside and outside the RBCs) from the 13C NMR spectral time courses. The Gárdos channel (also called KCa3.1 or KCNN4) mediates the efflux of K+ under the control of Ca2+. The membrane protein glycophorin is linked to the cytoskeleton, as is capnophorin; these linkages are made in as yet to be defined ways, but they are affected by Ca2+. Piezo1 mediates the exchange of both monovalent and divalent cations, with permeabilities (P) in the order of PK+ > PNa+PCs+ > PLi+PCa2+ (26). The free energy of covalent bond cleavage of glucose and its subsequent metabolites is captured as anhydride bond energy in ATP. This “energy currency” molecule is “spent” on driving the two ATPases shown in the diagram, as well as other kinase reactions not shown, producing adenosine diphosphate (ADP). Glycolysis is ATP demand–regulated so increased influx of Ca2+ stimulates Ca-ATPase that increases ATP hydrolysis and hence glycolytic flux. The positive feedback of extracellular lactate to Piezo1 is only speculated to occur in RBCs based on the findings on glomus cells from the carotid body (44).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/10/eaao1016/DC1

    Supplementary Discussion

    Supplementary Methods

    fig. S1. Control time course for the experiment in Fig. 1.

    fig. S2. Efflux of Cs+ from human RBCs suspended in gelatin gel.

    fig. S3. Influx of Cs+ to RBCs in gel-free suspension, in the presence of yoda1 at 37°C, measured with 133Cs NMR spectroscopy.

    table S1. Rates of [3-13C]l-lactate production by human RBCs in the absence and presence of the Piezo1 activator yoda1 and several effector reagents.

    References (5256)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Discussion
    • Supplementary Methods
    • fig. S1. Control time course for the experiment in Fig. 1.
    • fig. S2. Efflux of Cs+ from human RBCs suspended in gelatin gel.
    • fig. S3. Influx of Cs+ to RBCs in gel-free suspension, in the presence of yoda1 at 37°C, measured with 133Cs NMR spectroscopy.
    • table S1. Rates of 3-13CL-lactate production by human RBCs in the absence and presence of the Piezo1 activator yoda1 and several effector reagents.
    • References (52–56)

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