KMUP-1 inhibits pulmonary artery proliferation by targeting serotonin receptors/transporter and NO synthase, inactivating RhoA and suppressing AKT/ERK phosphorylation.

KMUP-1 inhibits monocrotaline (MCT)-induced pulmonary artery (PA) proliferation by targeting serotonin (5-HT) receptors, inactivating RhoA and reducing phosphorylation of AKT/ERK. In MCT-treated rats, KMUP-1 f (5 mg/kg p.o.; 1mg/kg i.p.x 21 days) decreased proliferation (PCNA-positive) cells and 5-HTT-expression in lung and 5-HT levels in plasma. In isolated PA, KMUP-1 and simvastatin (0.1-100 µM) inhibited 5-HT (10 µM)-induced PA constriction. l-NAME-pretreatment reduced KMUP-1-induced relaxation. In pulmonary arterial smooth muscle cells (PASMCs), KMUP-1 (1-100 µM) and simvastatin (10 µM) inhibited 5-HT-induced cell migration and proliferation and KMUP-1 (1-100 µM) inhibited 5-HT-induced Ca²+ influx. Similar to Y27632, KMUP-1 (1-100 µM) inhibited 5-HT-induced RhoA/ROCK expression, while KMUP-1, Y27632 and simvastatin at 10 µM inhibited 5-HT-induced 5-HTT expression and KMUP-1 inhibited 5-HT-induced phosphorylation of AKT and ERK1/2 in PASMCs. In human pulmonary arterial endothelial cell (HPAEC), KMUP-1 (1-100 µM) increased the expression of eNOS and 5-HT(2B) and also at 10 µM augmented eNOS expression and production of nitric oxide (NO) in 5-HT-treated HPAEC. In radioligand binding, the IC50/K(i) values of KMUP-1 for 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors were 0.34/0.0971, 0.04/0.0254, and 0.408/0.214 µM respectively. In conclusion, KMUP-1 inhibits MCT-induced PA proliferation by binding to 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors, increasing endothelial eNOS/5-HT(2B) receptor expression and NO release and inhibiting 5-HTT/RhoA/ROCK expression and AKT/ERK phosphorylation. KMUP-1 is suggested to be useful in the treatment of 5-HT-induced pulmonary artery proliferation.

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

BACKGROUND AND PURPOSE: KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats. EXPERIMENTAL APPROACH: We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats. KEY RESULTS: In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 microM) or the thromboxane A(2)-mimetic U46619 (0.5 microM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 microg x kg(-1) x min(-1), 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca(2+) efflux and angiotensin II-induced Ca(2+) influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression. CONCLUSION AND IMPLICATIONS: KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca(2+) desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH.

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel.

AIMS: This study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca(2+) sensitization and the K(+)-channel in chronic pulmonary arterial hypertension (PAH) rats. MAIN METHODS: Sprague-Dawley rats were divided into control, monocrotaline (MCT), and MCT+KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22. KEY FINDINGS: MCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 microM) and K(+) channel inhibitors (TEA, 10 mM; paxilline, 10 microM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT+KMUP-1-treated rats. By contrast, in beta-escin- or alpha-toxin-permeabilized PAs, CaCl(2)-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT+KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 microM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated K(+) channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT+KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K(+)-channel proteins to adapt to chronic PAH. SIGNIFICANCE: KMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca(2+) sensitivity and K(+)-channel function.

Inhibition of proinflammatory tumor necrosis factor-{alpha}-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.

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-alpha (TNF-alpha), a proinflammatory cytokine, increased the expression of inducible nitric-oxide synthase (iNOS) and NO production and decreased the expression of soluble guanylate cyclase alpha1 (sGCalpha1), soluble guanylate cyclase beta1 (sGCbeta1), protein kinase G (PKG), and the release of cGMP in TSMCs. The cell-permeable cGMP analog 8-Br-cGMP, xanthine-based KMUP-1 and KMUP-3, and the phosphodiesterase 5 inhibitor zaprinast all inhibited TNF-alpha-induced increases of iNOS expression and NO levels and reversed TNF-alpha-induced decreases of sGCalpha1, sGCbeta1, and PKG expression. These results imply that cGMP enhancers could have anti-proinflammatory potential in TSMCs. TNF-alpha also increased protein kinase A (PKA) expression and cAMP levels, cyclooxygenase-2 (COX-2) expression, and activated productions of prostaglandin (PG) E2 and 6-keto-PGF1alpha (stable PGI2 metabolite). Dexamethasone and N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398; a selective COX-2 inhibitor) attenuated TNF-alpha-induced expression of COX-2 and activated productions PGE2 and PGI2. However, KMUP-1 and KMUP-3 did not affect COX-2 activities and did not further enhance cAMP levels in the presence of TNF-alpha. It is suggested that TNF-alpha-induced increases of PKA expression and cAMP levels are mediated by releasing PGE2 and PGI2, the activation products of COX-2. In conclusion, xanthine-based KMUP-1 and KMUP-3 inhibit TNF-alpha-induced expression of iNOS in TSMCs, involving the sGC/cGMP/PKG expression pathway but without the involvement of COX-2.