Stimulation of mucin exocytosis from human epithelial cells by nitric oxide: evidence for a cGMP-dependent and a cGMP-independent pathway

The aim of this work was to investigate the role of nitric oxide (NO) on the macromolecular exocytotic function of human epithelial cells. We tested the effects of two NO-generating drugs, i.e. 1-hexanamine 6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl (MAHMA NONOate) and sodium nitroprusside (SNP), on mucin exocytosis from the human colonic epithelial HT29-Cl.16E cell line. Our results show that MAHMA NONOate and SNP elicited a rapid mucin exocytotic response through a cGMP-dependent and a cGMP-independent pathway respectively. Indeed, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a newly available specific inhibitor of soluble guanylate cyclase, inhibited both cGMP accumulation and subsequent mucin exocytosis evoked by MAHMA NONOate. By contrast, SNP did not alter intracellular cGMP levels, and SNP-mediated mucin exocytosis was not inhibited by ODQ. As expected from two NO donors acting through distinct pathways, the combined action of MAHMA NONOate and SNP led to an additive effect on mucin exocytosis. SNP was likely to act through S-nitrosylation of a cellular target, because cysteine, a reductive thiol that provides decoy targets for SNP through the formation of nitrosocysteine, abolished the early stimulatory effect of SNP on mucin exocytosis. Finally, the fact that in the presence of cysteine SNP was able to trigger a late, ODQ-inhibitable, mucin exocytotic response demonstrates the ability of NO to shift its intracellular signalling pathway depending on the changes of the redox state of the milieu.