1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7)has often been used in combination with protein kinase inhibitor (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to cellular processes, including the induction of gene expression. This use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Here we describe experiments that show that this interpretation is not correct with regard to the induction of two immediate-early genes, zif268 and c-fos, in PC12D cells. In these studies we confirmed that H7, but not HA1004, potently blocks the induction of zif268 and c-fos mRNA by nerve growth factor, carbachol, phorbol ester, Ca 2؉ ionophore, or forskolin. Surprisingly, however, H7 has no effect on the ability of these agents to activate mitogen-activated protein kinase ( In this study, we show that pretreating PC12D cells with H7, but not with HA1004, significantly reduces levels of phosphorylated RNA polymerase II in vivo. These results suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation.The immediate-early genes zif268 (also termed NGFI-A, egr-1, krox24, TIS8; reviewed in Ref. 1) and c-fos (2) encode transcription factors that have been proposed to function as "third messengers" in intracellular signal transduction cascades that convert information conveyed by extracellular stimuli into genomic responses that underlie growth, differentiation, and long term changes in the behavior of cells (3, 4). We have previously shown that NGF 1 (1) and the carbachol (carbamylcholine) cause the rapid induction of zif268 mRNA in PC12D cells (5). Induction of zif268 mRNA by NGF is mediated by the high affinity NGF receptor, TrkA, which activates the Ras/MAPK cascade (6). Induction by carbachol is mediated by the m1 subtype of muscarinic acetylcholine receptor, which activates phospholipase C to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (5). Increased intracellular levels of inositol 1,4,5-trisphosphate trigger the release of Ca 2ϩ from internal stores, which in turn opens "capacitative influx" Ca 2ϩ channels in the cell membrane, resulting in a sustained influx of extracellular Ca 2ϩ .2 Increased levels of diacylglycerol activate PKC. Both the sustained increase in intracellular Ca 2ϩ and the activation of PKC contribute to the induction of zif268 mRNA (5), at least in part by activating the MAPK cascade (81). Activation of the MAPK cascade is therefore a common element in the intracellular signaling events leading to gene expression that are initiated by NGF and carbachol in PC12D cells.In the course of investigating the involvement of PKC in t...