Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity protein kinase C in human peripheral T cells

The increase of intracellular free calcium concentration ([Ca2+](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 [Ca2+](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 muM, 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 Ca2+-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 muM, 20 min), (3) the acidification was dependent on the Ca2+ influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni2+, and (4) Thapsigargin (TG), a Ca2+-ATPase inhibitor, confirmed that acidification by the Ca2+ influx occurred in T cells in which PKC was not down-regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca2+ influx, exist in regulating pH(i) in T cells. This is the first report that PHA stimulates the acidification by Ca2+ 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. (C) 2001 Wiley-Liss, Inc.