Effect of cyclic GMP‐dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP

Abstract

1 In the present study we examined whether interleukin-lf (IL-Ifl) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-KB (NF-KB). In addition the interaction between cyclic AMP-and cyclic GMP-elevating agonists on the IL-iIf-stimulated expression of iNOS was examined.2 Exposure of vascular smooth muscle cells to IL-,IB stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-l, (60 u ml-')-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-' for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10-6 M for 2 min). 3 The IL-iI (60 u ml1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10-4 M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10-4 M). These compounds alone [rolipram (10-6 M), trequinsin (3 x 10-6 M) and Sp-8-CPT-cAMPS (l0-4 M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4 Inducible NOS protein was expressed in IL-/IB (30 u ml-', 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-,IB in combination with either rolipram (3 x 10-6 M) or Sp-8-CPTcAMPS (l0-4 M) but was reduced in those exposed to IL-,IB and Rp-8-CPT-cAMPS (l0-4 M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5 Exposure of smooth muscle cells to IL-lI (30 u ml-') for 30 min increased the level of NF-KB-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-KB-DNA complexes were found in cells which had been exposed to IL-,IB in combination with either Sp

Positive Feedback Regulation of Human Inducible Nitric-oxide Synthase Expression by Ras Protein S-Nitrosylation

“…In addition to regulating expression, NO is also able to attenuate iNOS activity by destabilizing iNOS dimers through effects on the heme prosthetic group or by S-nitrosylating iNOS directly (35). In contrast to negative regulation of iNOS by NO, studies have also established that NO can augment iNOS expression (37)(38)(39). Our findings support a role for iNOS-derived NO in the amplification of iNOS levels in human cells.…”

“…iNOS-derived NO can also inhibit iNOS enzymatic function through post-translational modifications (35,36). In contrast to negative regulation, NO positively regulates iNOS expression in certain rodent cell types and in human cells, but the mechanisms by which this positive feedback occurs is poorly understood (37)(38)(39)(40).…”

Cytokine-Stimulated Nitric Oxide Production in the Human Renal Proximal Tubule and Its Modulation by Natriuretic Peptides: A Novel Immunomodulatory Mechanism?

“…Although the human iNOS gene does not posses a sequence resembling the CRE consensus sequence [85], the influence of cAMP could still be an important factor in the transcriptional regulation of the iNOS gene [86]. Furthermore, the stimulatory action of cAMP does not appear to be related to the activation of NF-ĸB which is a nuclear transcription factor for iNOS [46, 87]. …”

Zaprinast, an inhibitor of cGMP‐selective phosphodiesterases, enhances the secretion of TNF‐α and IL‐1β and the expression of iNOS and MHC class II molecules in rat microglial cells

“…A recent study with mesangial cells suggested that NO-cGMP and cGMP analogues contribute to iNOS induction and its downregulation (Pérez-Sala et al, 2001). Though many other studies have presented the up or downregulation of iNOS expression by NO, these studies did not identify the cGMP-dependency of such regulations (Park et al, 1994;Mühl and Pfeilschifter, 1995;Boese et al, 1996;Park et al, 1997). Studies on iNOS expression have also shown different results with respect to up and downregulation.…”