Inhibition of Calpain Is a Component of Nitric Oxide-Induced Down-Regulation of Human Mast Cell Adhesion

Abstract

Nitric oxide is an important messenger that regulates mast cell activity by modifications to gene expression and intracellular pathways associated with exocytosis and adhesion. Integrin interactions with extracellular matrix components modulate an array of cell activities, including mediator production and secretion. To investigate the molecular mechanisms underlying NO regulation of mast cell function, we studied its effects on adhesion of a human mast cell line (HMC-1) to fibronectin (FN). The NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine strongly down-regulated the adhesion of HMC-1 to FN. Inhibitors of soluble guanylate cyclase and protein kinase G did not alter the response of cells to NO. A peroxynitrite scavenger did not affect modulation of adhesion by NO, nor could the effect of NO be mimicked by the peroxynitrite-producing compound 3-morpholinosydnonimine. NO donors inhibited the cysteine protease, calpain, while calpain inhibitors mimicked the effect of NO and led to a decrease in the ability of HMC-1 cells to adhere to FN. Thus, NO is an effective down-regulator of human mast cell adhesion. The mechanism for this action does not involve peroxynitrite or activation of soluble guanylate cyclase. Instead, a portion of NO-induced down-regulation of adhesion may be attributed to inhibition of the cysteine protease, calpain, an enzyme that has been associated with control of integrin activation in other cell types. The inhibition of calpain is most likely mediated via nitrosylation of its active site thiol group. Calpain may represent a novel therapeutic target for the regulation of mast cell activity in inflammatory disorders.

Nitric oxide: a major determinant of mast cell phenotype and function

“…Adhesion: activated MC are able to bind to extracellular matrix proteins, including fibronectin (FN). We have shown that treatment of the rat MC line RBL-2H3 with IFN-γ and HMC-1 with various NO donors abrogates the ability of both cell types to adhere to FN (Wills 1999, Forsythe & Befus 2003. In the latter case, the effects were mediated through the nitrosylation of the cysteine protease calpain in a cGMP-independent manner (Forsythe & Befus 2003).…”

“…In addition, we have demonstrated the expression of NOS isoforms and production of NO by various MC populations including rat peritoneal MC (PMC), human skin MC (HSMC) and human mast cell lines (HMC-1 and LAD-2) (Gilchrist et al 2002(Gilchrist et al , 2004. Furthermore, we investigated the role endogenous NO has on various MC functions including cytotoxicity, adhesion, early mediator release, and leukotriene production (Bissonnette et al 1991, Forsythe & Befus 2003, Gilchrist et al 2004.…”

Nitric Oxide Inhibits IgE-Dependent Cytokine Production and Fos and Jun Activation in Mast Cells

“…To date, there is little information available with regard to the mechanism of action of NO on mast cells, other than that guanylate cyclase and peroxynitrite are not involved (10,21) and that the cysteine protease calpain is a target in NO inhibition of mast cell adhesion (21). Particularly, the means by which NO is able to affect mast cell-dependent inflammation and the specific molecular mechanisms involved are incompletely understood.…”

“…For example, cell-or chemicalderived NO inhibits calcium ionophore-and IgE/Ag-induced release of granule-associated mediators from rodent peritoneal mast cells (9,10,19), adhesion of mast cells to fibronectin (20,21), as well as mast cell-mediated changes in gut epithelial permeability (22), adherence of leukocytes to the vascular endothelium (23), and microvascular leakage (24). Furthermore, the suppressive effect of LPS on mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity is mediated by NO (25).…”

Protein Tyrosine Nitration of Aldolase in Mast Cells: A Plausible Pathway in Nitric Oxide-Mediated Regulation of Mast Cell Function

“…SNOG is a well-known NO donor that favors a cGMP-independent mechanism of action of NO in different systems (28), and 500 mM SNOG has been widely used in different studies on MCs and inhibits MC function (6,(29)(30)(31). Moreover, SNOG has been used at up to 2 mM for 4 h to induce nitration in MCF-7 cells (32).…”