Inhibition of the calcium pump by high cytosolic Ca2+ in intact human red blood cells.

Pereira, Ana Cláudia; Samellas, D; Tiffert, Teresa; Lew, Virgilio L.

doi:10.1113/jphysiol.1993.sp019501

Cited by 12 publications

(11 citation statements)

References 25 publications

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“…The washed, ionophore-free packed RBCs (about 75% Hct) were resuspended (final Hct, about 14%) in 90K medium supplemented with inosine in a magnetically stirred glass vial in a water bath at 37°C; this immediately reactivated Ca 2+ extrusion 27 (t = 0) at the pump V max rate of each RBC. At 15- to 30-second intervals, 0.5 mL samples of this suspension were delivered into tubes containing 12 mL of 0K medium at 0°C to 4°C to instantly block further RBC Ca 2+ extrusion and trap their residual Ca 2+ content.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, the intrinsic measurement error of the reported Hb A1c values is within the size of the points in the figures below. Preliminary experiments (not shown) using Co 2+ instead of albumin washes 27 to terminate ionophore-mediated Ca 2+ fluxes 30 showed that Co 2+ alters the size and shape of the Hb A1c peaks, suggesting that Co 2+ might crosslink Hb with some other cell or lysate component that coeluted with genuine glycosylated Hb A1c. This precluded Co 2+ use when measuring Hb A1c.…”

Section: Methodsmentioning

confidence: 99%

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Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells

Lew

Daw

Etzion

et al. 2007

Blood

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Little is known about age-related changes in red blood cell (RBC) membrane transport and homeostasis. We investigated first whether the known large variation in plasma membrane Ca2+ (PMCA) pump activity was correlated with RBC age. Glycated hemoglobin, Hb A1c, was used as a reliable age marker for normal RBCs. We found an inverse correlation between PMCA strength and Hb A1c content, indicating that PMCA activity declines monotonically with RBC age. The previously described subpopulation of high-Na+, low-density RBCs had the highest Hb A1c levels, suggesting it represents a late homeostatic condition of senescent RBCs. Thus, the normal densification process of RBCs with age must undergo late reversal, requiring a membrane permeability increase with net NaCl gain exceeding KCl loss. Activation of a nonselective cation channel, Pcat, was considered the key link in this density reversal. Investigation of Pcat properties showed that its most powerful activator was increased intracellular Ca2+. Pcat was comparably selective to Na+, K+, choline, and N-methyl-D-glucamine, indicating a fairly large, poorly selective cation permeability pathway. Based on these observations, a working hypothesis is proposed to explain the mechanism of progressive RBC densification with age and of the late reversal to a low-density condition with altered ionic gradients.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells

Lew

Daw

Etzion

et al. 2007

Blood

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“…Four millilitres of this suspension were equilibrated in a tonometer at 37 0C for 10 min with either 02 or N2. Ionophore A23187 was added during this period to give a concentration of -100 ,umol (1 cells)-', which generates Ca2+ fluxes far above those of saturated Ca2`pumps and ensures a rapid and nearly total Ca2+ equilibration even in substrate-fed cells (Simonsen & Lew, 1980 (Pereira, Samellas, Tiffert & Lew, 1993). Another limitation is that medium and cell pH vary in parallel, preventing distinction of intra-and extracellular effects.…”

Section: Methodsmentioning

confidence: 99%

Effects of deoxygenation on active and passive Ca2+ transport and cytoplasmic Ca2+ buffering in normal human red cells.

Tiffert

Etzion

Bookchin

et al. 1993

The Journal of Physiology

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“…6 Ca2+ permeability of the membrane was about 10-4 cm S-1 [CaT]h load of 28 umol (340 g Hb)-'. Higher loads were avoided to prevent the inhibitory effects of high [CaT]i on the Ca2P pump Vmax (Pereira, Samellas, Tiffert & Lew, 1993). At t = 2 min, CoC12 in water was added from a 10 mm stock solution to give a final concentration of -40 uM in the suspension.…”

Section: Experimental Designmentioning

confidence: 99%

Cytoplasmic calcium buffers in intact human red cells.

Tiffert

Lew

1997

The Journal of Physiology

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1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their [Ca2+]1-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. (Lew, Tsien, Miner & Bookchin, 1982).This steady state is maintained with a minute total cell calcium content of 2-5 ,umol (1 cells)-', most of which could be extracted by external Ca2+ chelators from Ca2+-permeabilized cells (Harrison & Long, 1968;Bookchin & Lew, 1980;Engelmann & Duhm, 1987). Such small amounts of total and extractable Ca2P could be attributed to the scarcity of Ca2P-accumulating vesicles and to either the absence or unsaturation of cytoplasmic Ca2P buffers (Lew et al. 1982;Lew et al. 1985; Williamson, Puchulu, Penniston, Westerman & Schlegel, 1992 (Gardos, 1958;Tiffert, Spivak & Lew, 1988), binds to calmodulin (Scharif & Foder, 1978;Scharff, 1981), activates endogenous proteases and protease inhibitors (Glaser, Schwarz-Benmeir, Barnoy, Barak, Eshhar & Kosower, 1994;, promotes calpromotin binding to the cell membrane (Moore, Plishker & Shriver, 1990;Moore, Mankad, Shriver, Mankad & Plishker, 1991;Plishker, Chevalier, Seinsoth & Moore, 1992), and reacts with, or modifies the function of, a variety of other enzymes and cytoskeletal components (Lombardo & Low, 1994). Cytoplasmic Cah+ buffering in human red cells has been investigated in the past by a variety of methods, with similar overall results (Ferreira & Lew, 1976;Simons, 1982;Lew & Garcia-Sancho, 1989 In the analysis shown in Fig. 1 following usage, a will be treated as a dimensionless parameter. The true BAPTA concentrations were set at 5% (long-dash line) and 25% (short-dash line) below the nominal concentrations for both the sequential (Fig. 1A) and the simultaneous (Fig. 1B) protocols. In all cases, the mismatch generated false positive y-intercepts with apparent saturation patterns. In Fig. lA -s in human red cells 141BAPTA concentrations, respectively. In the simultaneous protocol (Fig. 1B) BAPTA was present, nominal BAPTA concentrations were used for the calculations. The conversion factor, f, used to minimize the difference between the plots with and without BAPTA, was then estimated as illustrated in Fig. 2A (A-D). BAPTA was not used in the last three experiments, in which the suspension media was pretreated with Chelex 100. The purity of BAPTA used for the remaining experiments in this set was 76%. The statistical parameters in each row correspond to the mean and standard errors derived from least mean squares fits to the two-buffer equation in each experiment (see Fig. 4). The...

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Inhibition of the calcium pump by high cytosolic Ca2+ in intact human red blood cells.

Cited by 12 publications

References 25 publications

Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells

Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells

Effects of deoxygenation on active and passive Ca2+ transport and cytoplasmic Ca2+ buffering in normal human red cells.

Cytoplasmic calcium buffers in intact human red cells.

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