Inhibition of Proinflammatory Tumor Necrosis Factor-α-Induced Inducible Nitric-Oxide Synthase by Xanthine-Based 7-[2-[4-(2-Chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-Nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) in Rat Trachea: The Involvement of Soluble Guanylate Cyclase and Protein Kinase G

Abstract

In the study of anti-proinflammation by 7-[2-[4-(2-chlorobenzene)piperazinyl] ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-3), exposure of rat tracheal smooth muscle cells (TSMCs) to tumor necrosis factor-␣ (TNF-␣), a proinflammatory cytokine, increased the expression of inducible nitric-oxide synthase (iNOS) and NO production and decreased the expression of soluble guanylate cyclase ␣ 1 (sGC␣ 1 ), soluble guanylate cyclase ␤ 1 (sGC␤ 1 ), protein kinase G (PKG), and the release of cGMP in TSMCs. The cell-permeable cGMP analog 8-BrcGMP, xanthine-based KMUP-1 and KMUP-3, and the phosphodiesterase 5 inhibitor zaprinast all inhibited TNF-␣-induced increases of iNOS expression and NO levels and reversed TNF-␣-induced decreases of sGC␣ 1 , sGC␤ 1 , and PKG expression. These results imply that cGMP enhancers could have anti-proinflammatory potential in TSMCs. TNF-␣ also increased protein kinase A (PKA) expression and cAMP levels, cyclooxygenase-2 (COX-2) expression, and activated productions of prostaglandin (PG) E 2 and 6-keto-PGF 1 ␣ (stable PGI 2 metabolite). Dexamethasone and N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398; a selective COX-2 inhibitor) attenuated TNF-␣-induced expression of COX-2 and activated productions PGE 2 and PGI 2 . However, KMUP-1 and KMUP-3 did not affect COX-2 activities and did not further enhance cAMP levels in the presence of TNF-␣. It is suggested that TNF-␣-induced increases of PKA expression and cAMP levels are mediated by releasing PGE 2 and PGI 2 , the activation products of COX-2. In conclusion, xanthine-based KMUP-1 and KMUP-3 inhibit TNF-␣-induced expression of iNOS in TSMCs, involving the sGC/cGMP/PKG expression pathway but without the involvement of COX-2.Pro-inflammatory cytokines, including TNF-␣, play an important role in regulating the tracheal smooth muscle contractility that is found in the asthmatic phenotype. TNF-␣ is increased in the sputa of patients with bronchial asthma and present in the bronchoalveolar lavage fluid of symptomatic asthmatic patients (Renauld, 2001). As a member of these cytokines, TNF-␣ attracts and activates nonspecific inflammatory macrophages and neutrophils during infection and hypersensitivity induced by the inhalation of organic particles or fumes (Mohr, 2004;Mendez-Samperio et al., 2006).Likewise, proinflammatory TNF-␣, inducible nitric oxideThis study was supported by grants

Liraglutide prevents and reverses monocrotaline-induced pulmonary arterial hypertension by suppressing ET-1 and enhancing eNOS/sGC/PKG pathways

“…To measure the protein expression levels, total proteins were extracted, and Western blotting analysis was performed as described previously3031. Mouse anti-eNOS monoclonal antibody 1:1000 (Transduction, N 30020, USA), rabbit anti-sGC polyclonal antiserum1:1000 (Cayman, 160890, USA), rabbit anti-PKG monoclonal antibody1:1000 (Calbiochem, 370652, USA), mouse anti-Rho kinase monoclonal antibody1:1000 (Upstate, 05–778, USA), and the loading control protein rabbit anti-GAPDH polyclonal antibody 1:1000 (Sigma, A5441, USA) and anti-β-actin polyclonal antibody 1:1000 (Sigma, A2066) were used.…”

The xanthine derivative KMUP‐1 inhibits models of pulmonary artery hypertension via increased NO and cGMP‐dependent inhibition of RhoA/Rho kinase

“…Whole right lung tissues of monocrotaline‐treated rats were isolated and cut into small chips to extract protein. To measure protein expression levels, the total proteins were extracted, and Western blotting analyses were performed as described previously (Wu et al ., 2006). Mouse or rabbit monoclonal antibodies to eNOS (Upstate, Lake Placid, NY, USA), sGCα (Sigma‐Aldrich, Saint Louis, CA, USA), sGCβ (UC, Santa Cruz, CA, USA), PDE‐5A (BD Tansduction, San Jose, CA, USA), PKG (Calbiochem, San Diego, CA, USA), ROCK II (Upstate), RhoA (UC), MYPT‐1 (Upstate), phospho‐MYPT‐1 (Thr‐696) (Upstate) and the loading control protein β‐actin (Sigma‐Aldrich, St. Louis, MO, USA) were used in the Western blot analyses.…”

“…Sildenafil, inhibitors of Rho kinase (ROCK) and statins have been used to treat PAH. Because the xanthine derivative KMUP‐1 (Figure 1A) raises cGMP levels (as does sildenafil), increases levels and activity of endothelial nitric oxide synthase (eNOS) and suppresses ROCK in smooth muscle (Wu et al ., 2006; Liu et al ., 2007), we postulated that this compound would ameliorate PAH.…”

KMUP-1 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss: Roles of MAPKs, Akt, NF-κB and Calcium/Calcineurin/NFATc1 Pathways

“…In addition, KMUP-1 has been shown to possess multifunctional properties, including anti-inflammatory [17], anti-proliferation [18], [19], cardioprotective [20] and neuroprotective properties [21]. However, the effects of KMUP-1 on osteoclast differentiation and bone resorption have not been reported.…”