Abstract

We have shown that neurokinin A-induced contraction of human sigmoid circular muscle (HSCM) is reduced in patients with ulcerative colitis and that interleukin (IL)-1β may play a role in this change. We now examine changes in the signal transduction pathway mediating neurokinin A-induced contraction of HSCM and explore the role of IL-1β and of H₂O₂ in these changes. In Fura 2-AM-loaded ulcerative colitis HSCM cells, neurokinin A- and caffeine-induced peak Ca²⁺ increase and cell shortening were significantly reduced. In normal cells, neurokinin A-induced contraction was decreased by protein kinase C inhibitor chelerythrine and by calmodulin inhibitor CGS9343B [1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate]. In ulcerative colitis muscle cells, contraction was inhibited only by chelerythrine but not by CGS9343B. IL-1β treatment of normal HSCM strips and cells reproduced the changes observed in ulcerative colitis. IL-1β-induced reduction in caffeine-induced peak Ca²⁺ increase and contraction was reversed by catalase, suggesting a role of H₂O₂. IL-1β-induced H₂O₂ production was inhibited by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (2′-amino-3′-methoxyflavone) and by cytosolic phospholipase A2 (cPLA₂) inhibitor AACOCF3 (arachidonyltrifluoromethyl ketone), but neither by p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] nor by nuclear factor-κB (NF-κB) inhibitory peptide NF-κB SN50 (H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). IL-1β significantly increased the phosphorylation of extracellular signal-regulated kinase 1 (ERK1)/ERK2 MAPKs and cPLA₂ and IL-1β-induced cPLA₂ phosphorylation was blocked by PD98059. We conclude that Ca²⁺ stores of HSCM cells may be reduced in ulcerative colitis and that the signal transduction pathway of neurokinin A-induced contraction switches from calmodulin- and protein kinase C-dependent in normal cells to protein kinase C-dependent in ulcerative colitis cells. IL-1β reproduces these changes, possibly by production of H₂O₂ via sequential activation of MAPKs (ERK1/ERK2) and cPLA₂.