Phorbol esters induce nitric oxide synthase and increase arginine influx in cultured peritoneal macrophages

Abstract

Incubation of peritoneal macrophages with /3-phorbol 12,13-dibutyrate promotes a time-dependent release of NO to the incubation medium. This effect was antagonized by LPS, a well known inducer of nitric oxide synthase (NOS) expression in macrophages, and was inhibited by .V"-methylt_-arginine and N"-nitro-L-arginine. An increase in intracellular cGMP and NOS activity was observed in parallel with NO release. The induction of NOS was accompanied by a stimulation of arginine influx within the cell. These results suggest that activation of protein kinase C by phorbol esters is sufficient to promote NOS induction in macrophages.Nitric oxide; Phorbol ester; Protein kinase C; Macrophage

Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells

“…This hypothesis is substantiated by our current findings, which demonstrate that, unlike LPS and IFN-γ-induction of NO synthesis, induction of -arginine transport is largely independent of PTKs. This process is also independent of PKC activation and contrasts with observations in rat peritoneal macrophages [29], human umbilical vein endothelial cells [30] and the human intestinal epithelial cell line Caco-2 [31], where PKC has been implicated as the key signalling mechanism mediating enhanced -arginine transport. Although there are cell, and maybe species, differences, it is worth noting that, in some of the studies mentioned above, PKC was implicated simply on results obtained with PKC activators including PMA, or on the use of relatively high concentrations of PKC inhibitors, which may not only be non-selective but also cytotoxic.…”

“…As with iNOS, the role of either PTK or PKC in enhanced -arginine transport induced by pro-inflammatory mediators remains inconclusive. Limited reports have suggested that exposure to phorbol esters enhances -arginine entry into rat peritoneal macrophages [29], human umbilical vein endothelial cells [30] and the human intestinal epithelial cell line Caco-2 [31]. However, Schmidlin and Wiesinger [7] failed to modulate arginine transport in activated astrocytes using the PKC inhibitor staurosporine.…”

Up-Regulation of Protein Kinase C-ϵ Promotes the Expression of Cytokine-inducible Nitric Oxide Synthase in RAW 264.7 Cells

“…1). Contrary to this behavior and in agreement with previous work (13,14), stimulation of primary cultures of rat peritoneal macrophages or isolated hepatocytes with PDBu produced a NO x Ϫ release that was 83 and 71%, respectively, of the response elicited after LPS treatment. This PDBu-dependent NO x Ϫ synthesis was absent in other cells such as isolated splenocytes, B cells, or human neutrophils.…”

“…The cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FCS, and antibiotics (50 g/ml of penicillin, streptomycin, and gentamicin). Peritoneal macrophages were obtained from Swiss mice following a published method (13). Rat hepatocytes, murine splenocytes and B cells, and human neutrophils were isolated and purified as described previously (14,26).…”

“…However, the existence of a phorbol ester-mediated iNOS induction mechanism has been observed in a more reduced number of cell types, as for example rat peritoneal macrophages and hepatocytes (13,14). In this regard, a role for PKC activation in the control of iNOS expression has been reported in the synergistic action of phorbol esters and IFN-␥ in murine peritoneal macrophages and in the cell line J774, as well as in astrocytes and microglia cells (15)(16)(17)(18).…”