Effect of GLC756, a novel mixed dopamine D1 receptor antagonist and dopamine D2 receptor agonist, on TNF-alpha release in vitro from activated rat mast cells
Introduction
Allergic eye disease is a common problem among individuals suffering from allergies (Bielory, 2000a, Bielory, 2000b, Part 1 and 2). Ocular allergic conjunctivitis is typically associated with IgE-mediated mast-cell activation in conjunctival tissue (Stahl et al., 2002). The binding of IgE to the high affinity receptor (FcεRI) on the surface of mast cells (MC) and basophils and its subsequent crosslinking with an allergen is the first step in a cascade of processes leading to the release of pro-inflammatory mediators (Shaikh et al., 1997). In addition to its crucial role in MC activation, IgE has been shown to regulate the FcεRI expression on the surface of MC (Welker et al., 1997; MacGlashan, 2005; Yamaguchi et al., 1997, Yamaguchi et al., 1999) and basophils (Lantz et al., 1997, MacGlashan et al., 1997). Therefore, increased IgE concentration induces a higher FcεRI density which in turn leads to an increased release of pro-inflammatory cytokines such as TNF-α (Brazis et al., 2002). Among the MC-derived pro-inflammatory mediators, TNF-α plays a prominent role. Therefore, compounds modifying TNF-α release from MC in response to allergen challenge may be of potential value for therapeutic intervention (Cook et al., 2000).
To investigate the potential of antiglaucoma medication to improve or possibly aggravate ocular allergic inflammation three topically active antiglaucoma drugs i.e. latanaprost, timolol and GLC756 which differ in their chemical structure and pharmacological profile were selected to examine their influence on TNF-α release from activated rat mast cells. Latanoprost, a prostaglandin F2α (PGF2α) analogue, is very effective in lowering elevated intra ocular pressure (IOP) by increasing uveoscleral outflow (Ziai et al., 1993, Alm and Stjernschantz, 1995). GLC756 a benzoquinoline derivative exhibits the profile of a mixed dopamine D1-receptor antagonist and dopamine D2 receptor agonist. It lowered IOP in man and experimental animals by an yet unknown mechanism (Pruente and Flammer, 1995) and in addition improved perfusion of the optic nerve in experimental animals (Prünte et al., 1995, Chiou and Chen, 1992, Markstein et al., 1996, Prunte et al., 1996). GLC 756 was assessed in this experiment since in a previous study it was found to decrease TNF-α levels in the serum of Lewis rats after intravenous injection of 160 μg lipopolysaccharide from Salmonella typhimurium (Laengle et al., 2006). Finally, timolol, a non-selective beta-receptor blocking agent which lowers IOP by decreasing aqueous humor inflow is a widely used antiglaucoma drug (Konstas et al., 2003). As positive control for the inhibition of TNF-α release the corticosteroid betamethasone, a potent anti-inflammatory drug (Xu et al., 2005), was used. In the present study, the effect of the test compounds was assessed in-vitro using a basophilic leukemia RBL-2H3 mast cell line. RBL-2H3 cells have been utilized by a number of laboratories as a model to study signal transduction mechanisms through the IgE pathway (Barsumian et al., 1981).
Section snippets
Drug preparation
Latanoprost was commercially available from Cayman Chemical (Ann Arbor, MI, USA) as a solution in methyl acetate, purity >98%. The compound was used as a solution in ethanol after evaporation of the methyl acetate under a stream of nitrogen. GLC756, as a powder, received from Novartis Pharma AG (Switzerland) was reported to be 100% pure by certificate of analysis. The drug was dissolved in DMSO (Sigma, Switzerland). Timolol was available commercially as a powder (100% pure) from Sigma,
Results
In the concentration range from 0.1–30 μM none of the test compounds produced an cytotoxic effect on RBL-2H3 cells (data not shown).
RBL-2H3 cells produced a high TNF-α release into the culture medium 5 h after activation with anti-IgE, based on a time-dependent TNF-α release between 20 min to 22 h post-activation (data not shown). The TNF-α values measured with the different control media were comparable. Therefore, the effect of the test compounds on TNF-α release was assessed at 5 h post
Discussion
Ocular allergic inflammation is a common problem among individuals suffering from allergies (Bielory, 2000a, Bielory, 2000b, Part 1 and 2). Activation of mast cells is the final step in allergic and pseudoallergic conditions resulting through the release of cytokines and other inflammatory mediators, in diseases such as allergic rhinitis and conjunctivitis (Smith et al., 2002, Church and McGill, 2002).
Especially, TNF-α release plays a crucial role by triggering other proinflammatory events in
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