Protein kinase C activation inhibits TCR-mediated calcium influx but not inositol trisphosphate production in HPB-ALL T cells.

The regulation by protein kinase C (PKC) of TCR-mediated changes in phosphoinositide metabolism and intracellular calcium ([Ca2+]i) was investigated in HPB-ALL T cells. Low concentrations (< 1 microgram/ml) of the anti-CD3 OKT3 mAb triggered large calcium signals but not detectable increase in D-myo-inositol 1,4,5-trisophate (IP3) production. CD3-CD4 coligation amplified the calcium signal twofold, compared with CD3 cross-linking alone, but this protocol also did not stimulate IP3 production. At higher OKT3 concentrations (> 2.5 micrograms/ml), IP3 production was detected but was not inhibited by activating PKC with phorbol ester. In contrast, PKC activation caused a marked inhibition (53 to 64%) of the CD3- or CD3-CD4-triggered calcium signals, but had only a small inhibitory effect (20 to 30%) on the release of intracellular Ca2+. PKC activation also inhibited by 47% calcium signals triggered by thapsigargin, an inhibition that was completely reversed by addition of the specific PKC inhibitor RO 31-8220 (1 microM). Addition of 1 microM RO 31-8220 caused a twofold stimulation of CD3-induced calcium signals. This effect was not mediated at the level of Ca2+ influx, because RO 31-8220 did not significantly increase thapsigargin-triggered calcium signals. However, RO 31-8220 did slightly increase the CD3-induced release of intracellular Ca2+, suggesting that amplification of Ca2+ influx may be secondary to increased release of Ca2+ from intracellular stores. Our results indicate that PKC regulates TCR-mediated changes in [Ca2+]i in HPB-ALL T cells by two distinct mechanisms. First, PKC activation causes a marked inhibition of Ca2+ influx by a mechanism independent of changes in IP3 production, possibly involving inhibition of ion channels. Second, PKC activity causes a small inhibition of intracellular Ca2+ release, most likely by promoting Ca2+ sequestration.