Neurokinin 1 receptors and neprilysin modulation of mouse bladder gene regulation

Neurokinin 1 (NK₁) receptors play a fundamental role in neurogenic inflammation. We sought to determine the mechanisms down-stream from NK₁ receptor (NK₁R) activation using cDNA arrays and a novel statistical method to analyze gene expression. We used female NK ₁R^-/- and wild-type (WT) mice that were sensitized actively by intraperitoneal injections of dinitrophenol 4 (DNP ₄)-human serum albumin. Cystitis was induced by intravesical instillation of antigen of DNP₄-ovalbumin, and control mice were challenged with saline. At 1, 4, and 24 h after instillation, bladders were removed for 1) RNA extraction (n = 3), 2) replicate of RNA extraction (n = 3), and 3) morphological analysis (n = 6). For cDNA array experiments, three bladders from each group were homogenized, and total RNA was obtained. DNase-treated RNA was reverse-transcribed to cDNA, labeled with [α- ³²P]dATP and hybridized to Atlas Mouse 1.2 Arrays (Clontech). After calculating the mean and SD for background spots, each experimental value was assigned a normalized score S using the formula S′ = (S - Av)/SD, where S′ is the original pixel value, and Av and SD are the mean and standard deviation of background spots, respectively. Only genes that expressed 3 SD values above background were used. Hypervariable genes were sorted by cluster analysis. Matrices of correlation coefficients were calculated and represented in a connectivity mosaic. As results, we found that in WT mice the most prominent gene cluster had neprilysin in a central position and positively correlated to a group of activator protein-1 (AP-1)-responsive genes, including laminin-α3, tissue plasminogen activator 11, fos-B, and TNF-β. In WT mice, antigen-induced bladder inflammation led to a downregulation in neprilysin expression. In contrast, NK₁R^-/- mice failed to mount an inflammatory reaction and presented neprilysin negatively correlated with the same genes described in WT. In conclusion, this work indicates an overriding participation of NK₁R and neprilysin in bladder inflammation, provides a working model for the involvement of AP-1 transcription factor, and evokes testable hypotheses regarding the role of NK₁R and neprilysin in inflammation.