Effects of protein kinase C (PK-C) activators and inhibitors on human large granular lymphocytes (LGL): Role of PK-C on natural killer (NK) activity

Abstract

The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10 −4–10 −5 g/ml) or with 12- O-tetradecanoylphorbol-13-acetate (TPA) (10 −8–10 −10 g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca 2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5–40 μ M), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 μ M), but not HA 1004 (50 μ M), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10 −6 g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.