Regulation of Free Intracellular Ca2+ in Resting and Con A-Stimulated Mouse Splenocytes
Free intracellular Ca2+ concentration f[Ca2+]i was measured using quin 2-loaded (Tsien et al., 1982a, b) mouse splenocytes. Resting cells had a f[Ca2+]i of 145.4 ± 48.9 nM. Addition of (5 µg/ml) concanavalin A (Con A), a polyclonal mitogen, to splenocyte culture doubled the f[Ca2+]i in about 5 min. Deprivation of external Ca2+ completely abolished this effect. In cells suspended in Na+-free buffer in absence of mitogen, a steady increase in f[Ca2+]i was observed suggesting the presence of a Na+/Ca2+ antiport system at the plasma membrane: the f[Ca2+]i doubled in Na+-free buffer in about 10 min. Addition of Con A to these cells resulted in a further two-fold increase in f[Ca2+]i. Addition of monensin (20 µM), a Na+ ionophore, also increased the basal f[Ca2+]i without affecting the Con A-stimulated increase. However ouabain (0.5 mM) had no effect on the basal Ca2+ levels during the short period in which it was tested. These results indicate that under physiological conditions, Na+/Ca2+antiport contributes to Ca2+ efflux and Con A stimulates Ca2+ influx thereby raising the f[Ca2+]i. Cells treated with pertussis toxin (50–450 ng/ml) did not show the usual increase in f[Ca2+]i upon addition of Con A, suggesting the involvement of Gi[or a similar GTP-binding protein, in Ca2+ mobilization.
KeywordsPertussis Toxin Bordetella Pertussis Plasma Membrane Vesicle Mouse Splenocytes Free Buffer
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