Changes in cytosolic free calcium concentration during myocardial ischemia were measured by 19F NMR in 5FBAPTA-loaded perfused rat hearts. The hearts were perfused with Krebs-Henseleit buffer containing 5 microM of the acetoxymethyl ester of 5FBAPTA, which was hydrolyzed by cytosolic esterases to achieve cytosolic concentrations of 5FBAPTA of 0.12 to 0.65 mM. Cytosolic free calcium concentrations were calculated as the product of the ratio of peak areas for bound and free 5FBAPTA in the NMR spectra and the dissociation constant (708 nM). The basal cytosolic calcium concentration, measured in potassium or magnesium arrested hearts, was 252 nM, and the time-average calcium concentration in beating hearts was 630 nM. Following the onset of total ischemia, there was no immediate substantial change in cytosolic calcium despite a rapid decline in creatine phosphate and ATP and a marked increase in inorganic phosphate as monitored by 31P NMR, but by 10 minutes, there was a substantial increase in free calcium concentration. The ratio of peak areas of bound and free 5FBAPTA returned to the preischemic value during reperfusion, and there was no detectable loss of 5FBAPTA from the heart. Creatine phosphate was also restored to its preischemic level during reperfusion. These results indicate that cytosolic free calcium increases during ischemia and is not immediately associated with lethal injury. This increase in cytosolic calcium may activate degradative enzymes that eventually could compromise myocyte viability.

Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart.

Nuclear magnetic resonance studies of cellular metabolism

To study the ionic basis of salt sensitivity in hypertension, 19F-, 13P-, and 23Na-nuclear magnetic resonance techniques were used to measure cytosolic free calcium (Cai), pH (pHi), free magnesium (Mgi), and sodium (Nai) in erythrocytes of essential hypertensive subjects (n = 19). Individuals were studied for 2 mo each on low- (UNaV 200 meq/d) salt diets, with the concomitant administration of nifedipine (10 mg t.i.d.) or placebo tablets for 1 mo of each diet. Salt loading elevated Cai and Nai while suppressing Mgi and pHi; these changes occurred predominantly in salt-sensitive subjects (n = 9). Nifedipine blunted the pressor response to salt loading > 50% (delta diastolic BP [high-low salt vs placebo] = 5 +/- 2 vs 14 +/- 2 mmHg, P < 0.05) and reversed salt-induced ionic changes, lowering Cai and elevating Mgi and pHi. Regardless of the definition of salt sensitivity, continuous relationships were observed between the pressure response to salt loading, the levels of Cai (r = 0.726, P < 0.001), Nai (r = 0.747, P < 0.001), and pHi (r = -0.754, P < 0.001), and the salt-induced change in Mgi (r = -0.757, P < 0.001). Altogether, these results emphasize the reciprocal and coordinate nature of intracellular ionic changes in response to dietary salt loading and calcium channel blockade in essential hypertension. They suggest that salt sensitivity is mediated by cellular calcium accumulation from the extracellular space, in association with magnesium depletion and acidification. Lastly, interpretation of intracellular ion measurements in the future will require concurrent assessment of dietary salt intake.

/pdf/intracellular-ionic-consequences-of-dietary-salt-loading-in-2nuxe3j06c.pdf

Intracellular ionic consequences of dietary salt loading in essential hypertension. Relation to blood pressure and effects of calcium channel blockade.

The calcium homeostasis of normal human red cells may appear deceptively simple Mature red cells lack Ca2+-accumulating organelles and Ca2+ signalling functions Their total calcium content and Ca2+ permeability (PCa) are extremely low, and they have minimal cytoplasmic Ca2+ buffering capacity compared to other cell types One property red cells share with all prokaryots and eukaryots is a low cytoplasmic ionized Ca2+ concentration ([Ca2*]i) The low [Ca2+]i represents the balance between passive Ca2+ influx and active Ca2+ extrusion Passive Ca2+ influx is through low capacity transport pathways with carrier properties (Ferreira and Lew 1977; Lew and Ferreira 1978; Desai et al 1991a) Active Ca2+ extrusion is mediated by a large-capacity (high-Vmax), ATP-fuelled P-type pump (PMCA), expressed with genomic variants 1 and 4 (Schatzmann 1966, 1982; Schatzmann and Vin-cenzi 1969; Rega and Garrahan 1986; Wang et al 1992; Carafoli 1992, 1997) Thus, as with the resting [Ca2+]i level in other cell types, the physiological low-[Ca2+]i condition of red cells is probably maintained within very narrow margins throughout their life-span (-120 days) There have been claims that the Ca2+ permeability, and hence [Ca2+]i, may increase in physiological conditions, transiently during shear stress in the microcirculation (Larsen et al 1981), or terminally, as part of red cell programmed death (Lew and Beauge 1979; Romero and Romero 1999a), and the evidence for and against will be discussed below

Calcium Homeostasis in Normal and Abnormal Human Red Cells

Coupled in vivo activity of creatine phosphokinase and the membrane-bound (Na+,K+)-ATPase in the resting and stimulated electric organ of the electric fish Narcine brasiliensis.

Red blood cells were loaded with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) by incubation with 50 microM of the acetoxymethyl ester (FBAPTA-AM), and cytosolic free Ca2+ was monitored with 19F-nuclear magnetic resonance (NMR). Loading with 50 microM FBAPTA-AM, which results in a final FBAPTA level of approximately 0.5 mM, caused only a 25-30% fall in cell ATP as measured by 31P-NMR when 5 mM pyruvate was present. Leakage of the NMR active Ca2+ indicator, which results from cell lysis, was corrected for with the addition of extracellular Eu3+ ions, extracellular ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), or washing. With this method, we have found basal levels of cytosolic free Ca2+ averaging 61 +/- 6 nM (means +/- SE, n = 19). When the intracellular level of FBAPTA was varied from 0.1 to 1.0 mM, there was no correlation between the level of cytosolic free Ca2+ and the level of loading with FBAPTA. Addition of 10 microM of the Ca2+ ionophore A23187 with extracellular Ca2+ set at different levels by Ca2+-EGTA buffers caused an increase in cytosolic free Ca2+ as expected. Furthermore, ATP depletion caused a two- to three-fold increase in cytosolic free Ca2+, consistent with inhibition of Ca2+ efflux via that Ca2+-ATPase.

Nuclear magnetic resonance measurement of cytosolic free calcium levels in human red blood cells.

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Nuclear magnetic resonance measurement of cytosolic free calcium levels in human red blood cells.