KINETICS OF CALCIUM-TRANSPORT ACROSS THE LYMPHOCYTE PLASMA-MEMBRANE

We have investigated plasma membrane Ca2+ transport by monitoring the fluorescence of human peripheral T-lymphocytes loaded with fura 2. Thapsigargin (TG) was utilized to block the Ca2+ -ATPase of the endoplasmic reticulum and elevate the cytosolic Ca2+ (Ca2+). Ca2+ influx was inhibited by chelating extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The rate of decline in the Ca(i)2+ signal of TG-treated lymphocytes after exposure to EGTA was used to assess Ca2+ extrusion across the plasma membrane. Initial rates of Ca2+ decline were examined in cells suspended in Na+-containing and Na+-free solutions; initial rates were linearly related to the [Ca2+] i at the onset of the Ca2+ decline and were unaffected by varying the extracellular Ca2+. Extracellular Na+ increased the rate of Ca2+ extrusion and decreased the threshold [Ca2+], for extrusion, indicating a substantial role for the Na+-Ca2+ exchange in Ca(i)2+ homeostasis. Both decreased temperature and calmodulin inhibition significantly slowed the Ca2+ decline in Na+-free HEPES-buffered solution, suggesting Ca2+ extrusion under these conditions was mediated by the Ca2+ pump. Protein kinase C (PKC) activation or inhibition did not affect the Ca(i)2+ decline parameters. However, Ca2+ accumulation and Mn2+ (a Ca2+ surrogate) uptake were significantly inhibited by activators of PKC. Cyclic nucleotides altered neither the parameters of the Ca(i)2+ decline nor Mn2+ uptake. Thus human T-lymphocytes exhibit Na+- and Ca2+ -dependent transporters characterized as the Na+-Ca2+ exchanger and Ca2+ pump. The main effect of PKC in these cells is the modulation of Ca2+ entry across the lymphocyte plasma membrane.