Cytoplasmic calcium buffers in intact human red cells

• Published in: The Journal of physiology Vol. 500; no. Pt 1; pp. 139 - 154
• Main Authors: Tiffert, T, Lew, V L
• Format: Journal Article
• Language: English
• Published: England The Physiological Society 01-04-1997
• Subjects: Adenosine Triphosphate - blood; Buffers; Calcimycin - pharmacology; Calcium - blood; Calcium-Transporting ATPases - antagonists & inhibitors; Calcium-Transporting ATPases - blood; Chelating Agents - pharmacology; Cytoplasm - metabolism; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Erythrocytes - drug effects; Erythrocytes - metabolism; Humans; In Vitro Techniques; Kinetics; Models, Chemical; Reproducibility of Results; Tetrathionic Acid - pharmacology; Vanadates - pharmacology
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.1997.sp022005

1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization
of their [Ca2+]i-dependent functions. This was investigated by using a refined method and experimental protocols which allowed
continuity in the estimates of [Ca2+]i, from nanomolar to millimolar concentrations, in the presence and absence of external
Ca2+ chelators. 2. The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting
the cells of ATP or by adding vanadate to the cell suspension. Cytoplasmic Ca2+ buffering was analysed from plots of total
cell calcium content vs. ionized cytoplasmic Ca2+ concentration ([CaT]i vs. [Ca2+]i) obtained from measurements of the equilibrium
distribution of 45Ca2+ at different external Ca2+ concentrations ([Ca2+]o), in conditions known to clamp cell volume and pH.
The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187. 3. The results showed the following.
(i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic
Ca2+ binding agent. (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to
0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16). This contrasts with
the variability previously reported. (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately
80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM)
whose possible identity is discussed. (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation
constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)). 4. Vanadate (1 mM)
inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7%. The cytoplasmic Ca2+ buffering behaviour in these cells
was similar to that found in ATP-depleted cells.