Abstract
The increase of intracellular free calcium concentration ([Ca^2+^]~i~) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pH~i~), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pH~i~ is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)‐stimulated T cells. T cells were isolated from human peripheral blood. The [Ca^2+^]~i~ and the pH~i~ were measured using, respectively, the fluorescent dyes, Fura‐2, and BCECF. In addition, down‐regulation of PKC activity by PMA (1 μM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC‐dependent and Ca^2+^‐independent, (2) in PKC down‐regulated T cells, PHA induced acidification; this effect was enhanced by pre‐treating the cells with the Na^+^/H^+^ exchange inhibitor, 5‐(N,N‐dimethyl)‐amiloride, (DMA, 10 μM, 20 min), (3) the acidification was dependent on the Ca^2+^ influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni^2+^, and (4) Thapsigargin (TG), a Ca^2+^‐ATPase inhibitor, confirmed that acidification by the Ca^2+^ influx occurred in T cells in which PKC was not down‐regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca^2+^ influx, exist in regulating pH~i~ in T cells. This is the first report that PHA stimulates the acidification by Ca^2+^ influx but not alkalinization in T cells after down‐regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA. J. Cell. Biochem. 81: 604–612, 2001. © 2001 Wiley‐Liss, Inc.