EFFECT OF CYCLIC GMP-DEPENDENT VASODILATORS ON THE EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN VASCULAR SMOOTH-MUSCLE CELLS - ROLE OF CYCLIC-AMP

1 In the present study we examined whether interleukin-1 beta (IL-1 be ta) increases the activity of adenylyl cyclase in vascular smooth musc le cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-kappa B (NF-kappa B). In addition the interaction between cyclic AMP- and cyclic GMP-elevating agonists on the IL-1 bet a-stimulated expression of iNOS was examined. 2 Exposure of vascular s mooth muscle cells to IL-1 beta stimulated, the formation of cyclic AM P 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. T his IL-1 beta (60 u ml(-1))-stimulated formation of cyclic AMP was mod est (about 3 fold at 60 u ml(-1) for 1 h) compared to that evoked by i soprenaline (about 9 fold at 3 x 10(-6) M for 2 min). 3 The IL-1 beta (60 u ml(-1) for 24 h)-stimulated accumulation of nitrite, which was t aken as an index of NO production, was concentration-dependently incre ased by preferential inhibitors of cyclic AMP-dependent phosphodiester ases (rolipram and trequinsin). This effect was reproduced by a specif ic 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 co mpounds alone [rolipram (10(-6) M), trequinsin (3 x 10(-6) M) and Sp-8 -CPT-cAMPS (10(-4) M)] slightly but significantly increased the releas e of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4 Indu cible NOS protein was expressed in IL-IP (30 u ml(-1), 24 h)-stimulate d smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which ha d been exposed to IL-1 beta in combination with either rolipram (3 x 1 0(-6) M) or Sp-8-CPT-cAMPS (10(-4) M) but was reduced in those exposed to IL-1 beta and Rp-8-CPT-cAMPS (10(-4) M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to ei ther Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS el icited no such effect. 5 Exposure of smooth muscle cells to IL-1 beta (30 u ml(-1)) for 30 min increased the level of NF-kappa B-DNA complex es in nuclear extracts as detected by electrophoretic mobility shift a ssay. Similar levels of NF-kappa B-DNA complexes were found in cells w hich had been exposed to IL-1 beta in combination with either Sp-8-CPT -cAMPS (10(-4) M), trequinsin (10(-5) M) or rolipram (10(-5) M). None of the modulators alone affected the basal level of NF-kappa B binding activity. 6 NO-donors [sodium nitroprusside (SNP) 10(-4) M; dinitrosy l-iron-di-L-cysteine-complex (DNIC), 10(-4) M; 3-morpholino-sydnonimin e (SIN-1), 10(-4) M] and atrial natriuretic factor (10(-6) M) signific antly increased the IL-1 beta (30 or 60 u ml(-1), 24 h)-stimulated exp ression of iNOS protein and activity as assessed indirectly by the con version of oxyhaemoglobin to methaemoglobin. In the absence of IL-1 be ta, SNP (10(-4) M, 24 h) but not the other cyclic GMP-dependent vasodi lators caused a modest expression of iNOS protein. No such effect was found in smooth muscle cells exposed to SNP in combination with Rp-8-C PT-cAMPS (10(-4) M) while an increased level of iNOS protein was Found in those exposed to SNP in combination with either Sp-8-CPT-cAMPS (10 (-4) M) or rolipram (3 x 10(-6) M). 7 Exposure of vascular smooth musc le cells to either S-nitroso-L-cysteine (Cys-SNO, 10(-4) M), SNP (10(- 4) M) or SIN-1 (10(-4) M) for 35 min affected minimally the basal acti vation of NF-kappa B but abolished that evoked by IL-1 beta (30 u ml(- 1) added during the last 30 min). However, addition of Cys-SNO followi ng the stimulation with IL-1 beta (during the last 5 min of the 30 min exposure period) reduced the level of NF-kappa B-DNA complexes only s lightly. 8 These data indicate that the cyclic AMP-dependent pathway p lays a decisive role in the signal transduction cascade initiated by t he activation of the IL-1 beta-receptor and leading to iNOS expression in vascular smooth muscle cells. The stimulatory effect of the cyclic AMP pathway on iNOS expression appears not to be related to the activ ation of NF-kappa B. In addition, cyclic GMP-dependent vasodilators po tentiate the cytokine-stimulated expression of iNOS probably by intera ction with the cyclic AMP effector pathway.