Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA170

Poster Communications

Cytoplasmic diffusivities of CO2 gas and H+ ions are reduced in diseases of human red cells that raise haemoglobin concentration

S. Richardson1, N. Roy2, P. Swietach1

1. Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, United Kingdom. 2. Department of Haematology and Oxford BRC Haematology Theme, Oxford University NHS Foundation Trust, Oxford, United Kingdom.

Background: Physiologically-adapted red blood cells (RBCs) must have adequate capacity to carry gases around the body and facilitate rapid gas exchange during their brief capillary transit time. A higher concentration of haemoglobin (Hb) will increase RBC gas carriage, but also produces a more tortuous environment for smaller molecules to diffuse in, which could restrict gas exchange. Our recent findings on wild-type RBCs from various animal species have demonstrated that higher Hb concentrations reduce the diffusivity of CO2 gas and H+ ions (1). The extent of this diffusive restriction can explain why RBCs with a higher mean corpuscular Hb concentration (MCHC) must also be thinner in order for gases to exchange rapidly and for cytoplasm to respond promptly to ambient changes in plasma pH (e.g. to execute the Bohr effect). Diseases of human RBCs are often linked to changes in cell shape and MCHC, which could have a hitherto unchartered effect on gas and H+ ion diffusion. Methods: Blood samples were obtained from hereditary spherocytosis (HS; n=3) and pyruvate kinase deficiency (PKD; n=1) patients (ethical approval 13-WA-0371), manifesting a raised (360, 358, 343 g/L) and reduced (299 g/L) MCHC, respectively, relative to control subjects (n=2; MHCH 330 g/L). RBCs were washed in EDTA buffer and settled in a superfusion chamber mounted on a confocal microscope. Diffusive properties in cytoplasm were interrogated by measuring the diffusive spread of H+ ions (reported with the fluorescent dye cSNARF1) in response to photolytic H+ uncaging (from 6-nitrobenzaldehyde; 1mM) applied to one end of the cell. The apparent H+ diffusion coefficient (DHapp) was measured in Hepes-buffered (CO2/HCO3--free) superfusates to probe Hb-facilitated H+ diffusion, and in 5% CO2/55 mM HCO3--buffered superfusates to estimate the additional facilitation by cytoplasmic CO2/HCO3-. Results: Hb-facilitated DHapp was slower in HS patients (4.4±0.4; 5.3±0.7; 4.2±0.4 µm2/s) and faster in the PKD patient (9.6±0.8 µm2/s), compared to wild-type RBCs. The addition of CO2/HCO3- buffer to cytoplasm produced a smaller acceleration of DHapp in HS cells (5.3±0.6; 5.9±0.5; 6.7±0.8 µm2/s) compared to PKD cells (12.8±1.3 µm2/s). Thus, a higher MCHC restricts Hb-assisted H+ diffusivity and impairs the ability of cytoplasmic CO2/HCO3- to shuttle H+ ions. The latter effect is attributable to reduced CO2 (and HCO3-) diffusivity in cytoplasm owing to Hb-dependent tortuosity. In combination with the increased diffusion distance arising from spherical geometry, RBCs from HS patients will have substantially slowed gas exchange which could contribute to reduced exercise tolerance. Physiological fitness of RBCs is thus a compromise between high gas-carrying capacity (favoured by high MCHC) and rapid cytoplasmic diffusivity (favoured by low MCHC).

Where applicable, experiments conform with Society ethical requirements