Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells

1 In RAW 264.7 murine macrophages and rat aortic smooth muscle (RASM) cells lipopolysaccharide (LPS) alone or in combination with interferon gamma (IFN gamma) or forskolin, respectively, stimulated the expression of the 130 kDa inducible isoform of nitric oxide synthase (iNOS) in both a time- and concentration-dependent manner. 2 Incubation with the direct activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) alone, did not result in detectable iNOS expression in either cell type. 3 Chronic PMA pretreatment resulted in significant down-regulation of alpha, beta and epsilon isoforms of PKC in RAW 264.7 macrophages and corresponded to a 20-30% reduction in LPS-induced iNOS expression. In contrast, IFN gamma alone or in combination with LPS stimulated an approximate 20% and 50% potentiation, respectively. 4 Pre-incubation with PKC inhibitors (calphostin C and H-7) showed similar effects upon stimulated induction of iNOS. 5 In RASM cells chronic PMA pretreatment resulted in down-regulation of alpha and epsilon PKC isoforms and corresponded to potentiation of iNOS expression in response to LPS alone or in combination with forskolin. 6 Co-incubation of RASM cells in the presence of PMA, angiotensin II (AII) or foetal calf serum (FCS) resulted in the inhibition of iNOS expression in response to LPS alone or in combination with forskolin. 7 Differential sensitivity to PKC inhibitors (calphostin C and H-7) was observed in RASM cells and exhibited both negative and positive modulation of stimulated induction. 8 In addition the PKC inhibitor compound Ro-31-8220 abolished stimulated induction in both cell types in response to all treatments. 9 These results suggest that PKC activation is required for induction of the 130 kDa isoform of NOS in both RAW 264.7 macrophages and RASM cells. However, individual PKC isoforms regulate iNOS expression in both a positive and negative manner.