Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (P TM ). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to P TM elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H 2 O 2 -sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47 phox or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with N-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H 2 O 2 , but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). ␣ 1 -Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H 2 O 2 constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H 2 O 2 , contribute to the myogenic response of arteriolar VSMCs. V ascular smooth muscle cells (VSMCs) of arterioles, but not arteries, are mechanosensitive, constricting to elevations in transmural pressure (P TM ). This myogenic response contributes to blood flow autoregulation and the establishment of basal vascular tone. 1 The response is an inherent property of arteriolar VSMCs involving calcium-dependent actin/myosin interaction, but the more proximal signaling components have not been clearly defined. 1 In cultured cells, mechanical stress initiates integrin-dependent activation of rho GTPases (rho, rac1, and CDC42), leading to reorganization of the cytoskeleton. 2,3 Cytoskeleton reorganization by rac1 is mediated by NADPH oxidase and generation of reactive oxygen species (ROS). 4 This enzyme complex, comprising Nox1, p47 phox , p67 phox , p22 phox , and rac1, is a key signaling system in cultured, noncontractile VSMCs. 5,6 The aim of the present study was to determine whether the rac1/NADPH oxidase/ROS signaling pathway regulates the myogenic response of arteriolar VSMCs. Materials and Methods Vasomotor ResponsesMouse-tail arterioles and arteries were cannulated in a microperfusion chamber (Living Systems) and studied in the absence of flow as described. 7 Unless stated otherwise, arterioles with intact endothelium were analyzed. Involvement of animals in the study was approved by the Ohio State University Animal Care and Use Committee. ROS DeterminationEndothelium-denuded vessels 7 were incubated with the H 2 O 2 -sensitive probe 5-(and 6)-chloromethyl-2Ј,7Ј-dichlorodihydro-fluorescein diacetate (DCF), 5 g/mL, for 30 minutes (37°C, P TM of 10 mm Hg). Because activation of DCF fluorescence is irreversible, fluorescent im...

Interferon (IFN)-Analysis of a genomic DNA clone for the human INDO gene identified an IFN-␥-responsive regulatory region upstream of the coding sequence by using reporter gene constructs and transfection assays (13,14). This IFN-␥-responsive region was narrowed down to a 67-base pair (bp) segment (Ϫ453 to Ϫ387) by deletion analyses (15). This 67-bp segment contains a number of potentially important sequence elements, which include a 14-bp sequence homologous to the ISRE sequence element (consensus sequence AGTTTCNNTTTCN(C/T), where N ϭ any nucleotide) found in IFN-␣-inducible genes and two 9-bp palindromic sequences (PE I and II) with homology to the GAS sequence (consensus TTNCNNNAA) found in IFN-␥-inducible genes (for a review, see Ref. 16) as shown in Fig. 1A, along with several other sequences of potential interest (a direct repeat and two inverted repeat sequence elements; not shown). Deletion and site-directed mutagenesis studies showed that IFN-␥-inducible expression directed by this regulatory region involved two sequence elements, the ISRE homolog and PE II palindromic sequence (15). Deletion or mutagenesis of either of these two elements essentially abolished the response to IFN-␥, indicating a complete dependence on these two sequence elements. The PE I element seemed inadequate in supporting the IFN-␥ response despite the homology to the GAS sequence (15).Studies that were carried out to identify the IFN-␥-regulated factors that interact with the two sequence elements showed that the ISRE homolog and the PE II element recognized IFN-␥-regulated factors IRF-1 and p91 (STAT1), respectively (15). It has been established that IFN-␥ induces the level of the IRF-1 factor (17), whereas p91 (STAT1) is activated by tyrosine phosphorylation of preexisting p91 protein through the involvement of JAK1 and JAK2 enzymes activated by IFN-␥ (16). We showed that expression of antisense to IRF-1 abolished the induction of the INDO gene by IFN-␥, indicating a complete dependence on IRF-1 factor (15). The involvement of IFN-␥-induced IRF-1 factor would explain earlier results that showed that IFN-␥-inducible expression of the INDO gene (then designated C5-4) required

Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)--induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation (90% of genes tested (33 of 37 genes). We conclude that our validated high-density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine-based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.