Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Intracellular trafficking in Plasmodium-infected erythrocytes

The past few years have witnessed considerable progress in molecular and biochemical studies of intracellular trafficking in malaria-infected red cells. Highlights include the identification of solute channels in the vacuolar membrane and the red blood cell membrane, a tubovesicular membrane network that delivers exogenous nutrients and drugs to the parasite, and parasite gene families that mediate adherence to endothelial cells and red cells.     

Intracellular distribution of DNA internalized through calcium phosphate precipitation

Although calcium phosphate precipitation is the most commonly used method for DNA-mediated gene transfer, the mechanism for its action is unknown. We showed recently that both transient and stable expression of exogenous genes in the transfected cells are entirely dependent on DNA internalized through active endocytosis. We now report on the subcellular distribution of the endocytosed DNA. After exposure to calcium phosphate-precipitated DNA, cultured fibroblasts internalized less than 10% of the DNA into the nuclei fraction. About 20% was recovered in each of the putative plasma membrane and vesicular organelle fractions. Although over 50% was recovered in the cytosolic fraction, it was completely degraded to oligonucleotides of smaller than 100 bp. In contrast, intact DNA molecules were recovered in all the other subcellular fractions. Similar patterns of DNA distribution were observed not only in the easily transformed mouse cells (Ltk-) but also in the transformation-resistant human primary fibroblasts. In conclusion, DNA-mediated gene transfer by calcium phosphate precipitation is an inefficient procedure because over 50% of the DNA is almost immediately degraded and released into the cytosol. Contrary to accepted views, DNA macromolecules do not seem to pass through the cytosol before reaching the nuclei. A novel transport pathway is proposed in which exogenous DNA molecules may be transferred directly by intermediary vesicles from the endocytic-lysosomal compartment to the nucleus.     

Intracellular calcium puffs in osteoclasts

We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.     

Intracellular calcium fluxes in human platelets

Fluorescence changes and secretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membrane-associated Ca2+. When extracellular [Ca2+] is decreased to less than 0.1 microM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilisation of membrane-associated Ca2+ on stimulation by maximal doses of five excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve. Similar EC50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence. The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilisation and Ca2+ removal can under some conditions involve sites which are not monitored by this probe.     

Intracellular calcium in cystic fibrosis heterozygotes

Increased intracellular calcium (Ca) has been reported in several cell types in cystic fibrosis (CF). Because CF is an autosomal recessive trait examination of asymptomatic obligate carriers (HZ) of the gene is a powerful way to determine the relevance of this observation to the abnormal gene product. We report here that Ca as determined by atomic absorption spectrophotometry in cultured skin fibroblasts and circulating lymphocytes is greater in HZ than in control cells. Since an intracellular Ca increase is expressed in HZ the Ca differences in CF likely reflect action of the gene product responsible for CF and not some secondary or tertiary effect of the disease.     

Amino acid transport by resealed ghosts from pigeon erythrocytes.

Resealed ghosts from pigeon erythrocytes were shown to haemolyse during incubation in isotonic media with pH values greater than about 7 and high concentrations of Na+ inside the ghosts seemed to enhance this effect. At lower pH values the ghosts were stable but still highly permeable to Na+ and K+, and moderately permeable to sucrose. Under the latter conditions the ghosts transported amino acids in a way qualitatively but not quantitatively similar to intact erythrocytes. The Na+-dependent transport of serine and alanine by the ghosts consisted essentially of an exchange of extracellular for intracellular amino acids, with no significant net flux. In contrast, net fluxes of glycine in the direction of the Na+-concentration gradient across the ghost membrane were demonstrated. However, under one condition a small net influx of glycine occurred against the prevailing Na+-concentration gradient. Unlike Na+-dependent glycine uptake, the uptake of six other amino acids by intact pigeon erythrocytes was not influenced by the nature of the anion present. The significance of these findings in relation to previous work on the Na+-gradient hypothesis of membrane transport is discussed.     

Synthesis of nicotinamide adenine dinucleotide in the nuclei of pigeon erythrocytes]

The nuclei of pigeon erythrocytes are capable of synthesizing NAD from nicotinamid-mononucleotides and ATP. Some data on the kinetics of NAD-pyrophosphorylase have been obtained: the optimal concentration of nuclei and the effect of various incubation time. The pretreatment of nuclei by Triton X-100, or by ultrasonics enhances NAD synthesis. The results suggest that cyclic 3',5'-AMP (Fluka) may have no effect on NAD synthesis. The control of the cell metabolism by NAD formation is considered.     

Arachidonic acid increases intracellular calcium in erythrocytes

Recently, we have measured in erythrocytes a voltage-modulated and dihydropyridine-inhibited calcium influx. Since arachidonic acid and other polyunsaturated fatty acids influence the activities of most ion channels, we studied their effects on the erythrocyte Ca(2+) influx. It was measured on fresh erythrocytes, isolated from healthy donors, using the fluorescent dye Fura 2 as indicator of [Ca(2+)](i). AA (5-50 microM) and EPA (20-30 microM) stimulated a concentration-dependent increase in [Ca(2+)](i), deriving from extracellular calcium (1 mM), without affecting the intra- and extracellular pH and membrane voltage. The Ca(2+) influx rate varied from 0.5 to 3 nM Ca(2+)/s in the presence of AA and from 0.9 to 1.7 nM Ca(2+)/s with EPA. The Ca(2+) influx elicited by AA and EPA was not inhibited by dihydropyridines, while cyclooxygenase inhibitors were effective and PGE1 or PGE2 did not produce any effect. We conclude that AA could activate an erythrocyte voltage-independent Ca(2+) transport via an intermediate product of cyclooxygenase pathway; however, a direct interaction with the membrane lipid-protein cannot be excluded.