Paul Forsythe

Mast cells and nitric oxide: control of production, mechanisms of response.

Mast cells are involved in numerous activities ranging from control of the vasculature, to tissue injury and repair, allergic inflammation and host defences. They synthesize and secrete a variety of mediators, activating and modulating the functions of nearby cells and initiating complex physiological changes. Interestingly, NO produced by mast cells and/or other cells in the microenvironment appears to regulate these diverse roles. This review outlines some of the pathways central to the production of NO by mast cells and identifies many of the tightly controlled regulatory mechanisms involved. Several cofactors and regulatory elements are involved in NO production, and these act at transcriptional and post-translational sites. Their involvement in NO production will be outlined and the possibility that these pathways are critically important in mast cell functions will be discussed. The effects of NO on mast cell functions such as adhesion, activation and mediator secretion will be examined with a focus on molecular mechanisms by which NO modifies intracellular signalling pathways dependent or independent of cGMP and soluble guanylate cyclase. The possibility that NO regulates mast cell function through effects on selected ion channels will be discussed. Metabolic products of NO including peroxynitrite and other reactive species may be the critical elements that affect the actions of NO on mast cell functions. Further understanding of the actions of NO on mast cell activities may uncover novel strategies to modulate inflammatory conditions.

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

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.

Autonomic nervous system regulates secretion of anti-inflammatory prohormone SMR1 from rat salivary glands

The autonomic nervous system regulates the secretion of bioactive proteins and peptides from salivary glands that can be important in systemic physiological responses. The prohormone submandibular rat-1, which is highly expressed in rat submandibular glands, can be cleaved to produce polypeptides with analgesic and anti-inflammatory activities. Human genes related to submandibular rat-1 have conserved biological functions and are potentially important in pain suppression, erectile function, and inflammation. In this study we describe the differential expression and posttranslational modification of submandibular rat-1 protein in salivary glands, the urogenital tract, lung, blood, and saliva in male Sprague-Dawley and Brown Norway rats. Submandibular rat-1 protein is secreted into saliva after the administration of beta-adrenergic or cholinergic agonists. Removal of the sympathetic ganglion that innervates the salivary glands results in increased levels of submandibular rat-1 protein in salivary glands. The secretion of submandibular rat-1 in response to physiological stress may provide a large pool of submandibular rat-1-derived peptide products that can promote analgesia and decrease inflammation locally and systemically. This pathway may be conserved among mammals and may constitute an important anti-inflammatory and analgesic response to stress.

Immunomodulation by the submandibular gland

Abstract We have established that decentralization (cutting sympathetic nerve trunk) of the superior cervical ganglia bilaterally reduces the magnitude of allergic inflammation in the airways of rats. The magnitude of anaphylactic and endotoxic hypotension, and of gastrointestinal inflammation was also reduced. This anti-inflammatory activity was dependent upon intact submandibular glands. Reconstitution of sialadenectomized (removal of submandibular glands) rats with soluble extracts of the submandibular glands identified two polypeptides with anti-inflammatory activities. The sequences of these polypeptides were found within a prohormone, submandibular gland rat 1 (SMR1). The C-terminal peptide TDIFEGG, has been studied extensively. Using sequential amino acid substitutions and systematic removal of C terminal or N terminal amino acids, we established that the tripeptide FEG is biologically active. Modification of FEG to the D-isomeric feG, enhances its activity in some assay systems. We postulated that feG would inhibit airways inflammation, and tested this using a model ofallergic asthma, namely the Brown Norway rat sensitized to ovalbumin (OA). Sensitized rats were challenged 14 to 21 days later with aerosolized OA. This challenge markedly increased numbers of inflammatory cells recovered from the airways after 24 hour (29×10 6 , n=23) compared to saline controls (1×10 6 , n=4). The infiltrating cells included macrophages (10×10 6 ), neutrophils (9×10 6 ) and eosinophils (9×10 6 ). Intravenous (0.25 mg/kg) or oral feG (1 mg/kg) given 30 min prior to OA significantly inhibited influx of inflammatory cells by 50 to 70%. feG reduced inflammatory cell infiltration when given 30 min before to 3 to 6 hour post allergen exposure. Oral feG reduced the numbers of macrophages, neutrophils and eosinophils. One of the mechanisms underlying the effects of feG may be its ability to inhibit PAF-induced expression of CD11b on purified human neutrophils. It is possible that feG may be useful in the treatment of allergic asthma, given either as an oral prophylactic, or as a post exposure therapeutic. The cervical sympathetic nerve trunk-submandibular gland axis of neuroendocrine regulation of inflammation may be dysfunctional in inflammatory diseases and provide opportunities for new therapeutic intervention. This axis may be sensitive to modulation by central and peripheral neural mechanisms that influence its function/dysfunction.