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 the block the Ca(2+)-ATPase of the endoplasmic reticulum and elevate the cytosolic Ca2+ (Ca2+i). 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 Ca2+i signal of TG-treated lymphocytes after exposure to EGTA was used to assess Ca2+ extrusion across the plasma membrane. Initial rates of Ca2+i 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+i decline and were unaffected by varying the extracellular Ca2+. Extracellular Na+ increased the rate of Ca2+ extrusion and decreased the threshold [Ca2+]i for extrusion, indicating a substantial role for the Na(+)-Ca2+ exchange in Ca2+i homeostasis. Both decreased temperature and calmodulin inhibition significantly slowed the Ca2+i 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 Ca2+i decline parameters. However, Ca2+ accumulation and Mn2+ (a Ca2+ surrogate) uptake were significantly and Mn2+ (a Ca2+ surrogate) uptake were significantly inhibited by activators of PKC. Cyclic nucleotides altered neither the parameters of the Ca2+i decline nor Mn2+ uptake. Thus human T-lymphocytes exhibit Na(+)- and Ca(2+)-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.
Balasubramanyam
M
M
Hypertension Research Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103-2714.
Kimura
M
M
Aviv
A
A
Gardner
J P
JP
D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007474:Ion Exchange; D016623:Ion Pumps; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000494:pharmacology; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism
D016623:Ion Pumps; D009712:Nucleotides, Cyclic; D012964:Sodium; D011493:Protein Kinase C; D002118:Calcium