INVESTIGATION OF THE ROLE OF NITRIC-OXIDE AND CYCLIC-GMP IN BOTH THE ACTIVATION AND INHIBITION OF HUMAN NEUTROPHILS

1 The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GM P pathways. In addition, inhibition of neutrophil chemotaxis by NO rel easing compounds and increases in neutrophil nitrate/nitrite and cycli c GMP levels were examined. The ultimate aim of this work was to resol ve the paradox that NO both activates and inhibits human neutrophils. 2 A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalani ne (fMLP)-induced chemotaxis was supported by the finding that the NO synthase (NOS) inhibitor L-NMMA (500 mu M) inhibited chemotaxis; EC50 for fMLP 28.76 +/- 5.62 and 41.13 +/- 4.77 pmol/10(6) cells with and w ithout L- NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 mu M) inhibited chemotaxis; EC50 for fMLP 19.71 +/- 4.23 and 31.6 8 +/- 8.50 pmol/10(6) cells with and without carboxy-PTIO, respectivel y. 3 A role for cyclic GMP as a mediator of chemotaxis was supported b y the finding that the guanylyl cyclase inhibitor LY 83583 (100 mu M) completely inhibited chemotaxis and suppressed the maximal response; E C50 for fMLP 32.53 +/- 11.18 and 85.21 +/- 15.14 pmol/10(6) cells with and without LY 83583, respectively. The same pattern of inhibition wa s observed with the G-kinase inhibitor KT 5823 (10 mu M); EC50, for fM LP 32.16 +/- 11.35 and > 135 pmol/10(6) cells with and without KT 5823 , respectively. 4 The phosphatase inhibitor, 2,3-diphosphoglyceric aci d (DPG) (100 mu M) which inhibits phospholipase D, attenuated fMLP-ind uced chemotaxis; EC50 for fMLP 19.15 +/- 4.36 and 61.52 +/- 16.2 pmol/ 10(6) cells with and without DPG, respectively. 5 Although the NOS inh ibitors L-NMMA and L-canavanine (500 mu M) failed to inhibit fMLP-indu ced SAG, carboxy-PTIO caused significant inhibition (EC50 for fMLP 36. 15 +/- 7.43 and 86.31 +/- 14.06 nM and reduced the maximal response fr om 22.14 +/- 1.5 to 9.8 +/- 1.6 nmol O-2(-)/10(6) cells/10 min with an d without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6 A role for cyclic GMP as a mediator of SAG was supported by the effects of G-kinase inhibitors KT 5823 (10 mu M) and Rp-8-pCPT-cGMPS (100 mu M) which inhibited SAG giving EC50 for fMLP of 36.26 +/- 8.77 and 200.01 +/- 43.26 nM with and without KT 5823, and 28.35 +/- 10.8 and 49.25 +/- 16.79 nM with and without Rp-8-pCTP-cGMPS . 7 The phosphatase inhibitor DPG (500 mu M) inhibited SAG, EC50 for f MLP 33.93 +/- 4.23 and 61.12 +/- 14.43 nM with and without DPG, respec tively. 8 The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC50 = 14.72 +/- 1.6 mu M) > GEA 5024 (IC50 = 18.44 +/- 0.43 mu M) > SIN-1 (IC50 > 1000 mu M). Th is order of potency correlated with their ability to increase cyclic G MP levels rather than the release of NO, where SIN-I was most effectiv e (SIN-1 (EC50 = 37.62 +/- 0.9 mu M) > GEA 3162 (EC50 = 39.7 +/- 0.53 mu M) > GEA 5024 (EC50 = 89.86 +/- 1.62 mu M)). 9 In conclusion, chemo taxis and SAG induced by fMLP can be attenuated by inhibitors of phosp holipase D, NO and cyclic GMP, suggesting a role for these agents in n eutrophil activation. However, the increases in cyclic GMP and NO indu ced by fMLP, which are associated with neutrophil activation, are very small. In contrast much larger increases in NO and cyclic GMP, as obs erved with NO releasing compounds, inhibit chemotaxis.