Abstract
We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.
Highlights
We have studied the effectsof extracellular nucleo- natural killer cell cytotoxicity [11, 12], phosphoinositide hytides on the cytosolic free calcium concentration drolysis [13, 14], alterations in Fc receptor-mediated phago
The sustained peak Il elevation had two components, a “low threshold” (1 p~ ATP) response which saturated at10-50 p~ ATP and a“high threshold” response, apparent at[ATP] > l o 0 p ~ T.he latter component was not seen with nucleotides other than ATP and correlated withan ATP-induced generalized increase inplasma membrane permeability
Dahlquist and Diamant [28] and Cockcroft and Gomperts [29]have shown that, inmast cells, ATP- mediates histamine secretion, a process dependent on extracellular Ca2+.We have shown that ATP” increases plasma membrane permeability to ffiRb+ in5774 cells,a mouse macrophage-like cell line [30]
Summary
Probenecid was added at a final concentration of2.5mM. Control experiments showed that 2.5 mM probenecid does not affect nucleotide-mediated changes in [Ca2+Ii n 5774 cells, it markedly improves loading efficiency. Extracellular ATP Induces Calcium Fluxesfrom Both Internal Stores and across Plasma Membrane-The average resting cytosolic Ca2+concentration for 5774 cells loaded with quin was 115 nM (range of 75-150 nM); for cells loaded with indo, it was 160 nM (range of 130-210 nM). Bisozonol Loading-Cells were suspended in standard saline con- under "Experimental Procedures." The tracings on the left represent taining 1mMCaC12 and 1 mMMgC12 (pH 7.45) (final cell concentra- experiments performed in standard saline containing 1mM M P and tion of 2 X lo6cells/ml). To this suspension, 200 nM bisoxonol was 1mM Ca2+;the tracings on the right represent experimentsperformed in the presence of 5 mM EGTA.
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