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Xanthine-based KMUP-1 was shown to inhibit phosphodiesterases (PDEs) and modulate G-protein coupled receptors (GPCRs) to lower hyperlipidemia and body weight. This study further investigated whether KMUP-1 affects adipogenesis and lipolysis in 3T3-L1 preadipocytes. KMUP-1 (1⁻40 µM) concentration-dependently attenuated Oil Red O (ORO) staining and decreased triglyceride (TG) accumulation, indicating adipogenesis inhibition in 3T3-L1 cells. In contrast, the β-agonist ractopamine increased ORO staining and TG accumulation and adipogenesis. KMUP-1 (1⁻40 µM) also reduced MAPKs/Akt/PPARγ expression, PPARγ1/PPARγ2 mRNA, and p-ERK immunoreactivity at the adipogenesis stage, but enhanced hormone sensitive lipase (HSL) immunoreactivity at the lipolysis stage. Addition of protein kinase A (PKA) or protein kinase G (PKG) antagonist (KT5720 or KT5728) to adipocytes did not affect HSL immunoreactivity. However, KMUP-1 did increase HSL immunoreactivity and the effect was reduced by PKA or PKG antagonist. Simvastatin, theophylline, caffeine, and sildenafil, like KMUP-1, also enhanced HSL immunoreactivity. Phosphorylated HSL (p-HSL) was enhanced by KMUP-1, indicating increased lipolysis in mature 3T3-L1 adipocytes. Decreases of MAPKs/Akt/PPARγ during adipogenesis contributed to inhibition of adipocyte differentiation, and increases of PKA/PKG at lipolysis contributed to HSL activation and TG hydrolysis. Taken together, the data suggest that KMUP-1 can inhibit hyperadiposity in 3T3-L1 adipocytes.

We have previously demonstrated that KMUP-1, a xanthine-based nitric oxide enhancer, attenuates diabetic glomerulosclerosis, while increasing renal endothelial nitric oxide synthase expression in rats. However, the anti‑fibrotic mechanisms of KMUP‑1 treatment in diabetic nephropathy in terms of cell biology and transforming growth factor-β1 (TGF‑β1) remain unclear. Therefore, the present study involved investigating the effects of KMUP‑1 on high glucose (HG) or TGF‑β1‑induced pro‑fibrotic proteins in mouse mesangial (MES13) cells, and the effects of KMUP‑1 on streptozotocin (STZ)‑induced diabetic rats. It was identified that KMUP‑1 (10 µM) attenuated HG (30 mM)‑induced cell hypertrophy while attenuating TGF‑β1 gene transcription and bioactivity in MES13 cells. In addition, KMUP‑1 attenuated TGF‑β1 (5 ng/ml)‑induced Smad2/3 phosphorylation while attenuating HG or TGF‑β1‑induced collagen IV and fibronectin protein expression. Furthermore, KMUP‑1 attenuated HG‑decreased Suv39h1 and H3K9me3 levels. Finally, KMUP‑1 attenuated diabetes-induced collagen IV and fibronectin protein expression in STZ‑diabetic rats at 8 weeks. In conclusion, KMUP‑1 attenuates HG and TGF‑β1‑induced pro‑fibrotic proteins in mesangial cells and attenuation of TGF‑β1‑induced signaling and attenuation of HG‑decreased Suv39h1 expression may be two of the anti-fibrotic mechanisms of KMUP‑1.

We have demonstrated that KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) blunts monocrotaline-induced pulmonary arterial hypertension by altering Ca 2+ sensitivity, K + -channel function, endothelial nitric oxide synthase activity, and RhoA/Rho kinase (ROCK) expression. This study further investigated whether KMUP-1 impedes uridine 5'-triphosphate (UTP)-inhibited delayed rectifying K + (K DR ) current in rat pulmonary arteries involved the RhoA/ROCK signaling. Pulmonary artery smooth muscle cells (PASMCs) were enzymatically dissociated from rat pulmonary arteries. KMUP-1 (30μM) attenuated UTP (30μM)-mediated membrane depolarization and abolished UTP-enhanced cytosolic Ca 2+ concentration. Whole-cell patch-clamp electrophysiology was used to monitor K DR currents. A voltage-dependent K DR current was isolated and shown to consist of a 4-aminopyridine (5mM)-sensitive component and an insensitive component. The 4-aminopyridine sensitive K DR current was suppressed by UTP (30μM). The ROCK inhibitor Y27632 (30μM) abolished the ability of UTP to inhibit the K DR current. Like Y27632, KMUP-1 (30μM) similarly abolished UTP-inhibited K DR currents. Superfused protein kinase A and protein kinase G inhibitors (KT5720, 300nM and KT5823, 300nM) did not affect UTP-inhibited K DR currents, but the currents were restored by adding KMUP-1 (30μM) to the superfusate. KMUP-1 reversal of K DR current inhibition by UTP predominantly involves the ROCK inhibition. The results indicate that the RhoA/ROCK signaling pathway plays a key role in eliciting PASMCs depolarization caused by UTP, which would result in pulmonary artery constriction. KMUP-1 blocks UTP-mediated PASMCs depolarization, suggesting that it would prevent abnormal pulmonary vasoconstriction., (Copyright © 2016. Published by Elsevier Taiwan.)

KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) has been reported to cause hepatic fat loss. However, the action mechanisms of KMUP-1 in obesity-induced steatohepatitis remains unclear. This study elucidated the steatohepatitis via matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor α (TNFα), and related lipolysis via hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) by KMUP-1. KMUP-1 on steatohepatitis-associated HSL/p-HSL/ATGL/MMP-9/TNFα/interleukin-10 (IL-10) and infiltration of M1/M2 macrophages in obese mice were examined. KMUP-1 was administered by oral gavage from weeks 1-14 in high-fat diet (HFD)-supplemented C57BL/6J male mice (protection group) and from weeks 8-14, for 6 weeks, in HFD-induced obese mice (treatment group). Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining of tissues, oil globules number and size, infiltration and switching of M1/M2 macrophages were measured to determine the effects on livers. IL-10 and MMP-9 proteins were explored to determine the effects of KMUP-1 on M1/M2 macrophage polarization in HFD-induced steatohepatitis. Long-term administration of KMUP-1 reversed HFD-fed mice increased in body weight, sGOT/sGPT, triglyceride (TG) and glucose. Additionally, KMUP-1 decreased MMP-9 and reactive oxygen species (ROS), and increased HSL/p-HSL and IL-10 in HFD mice livers. In conclusion, KMUP-1, a phosphodiesterase inhibitor (PDEI), was shown to reduce lipid accumulation in liver tissues, suggesting that it could be able to prevent or treat steatohepatitis induced by HFD.

Objectives: Endothelial damage is strongly associated with cardiovascular diseases such as atherosclerosis, thrombosis and hypertension. Endothelial progenitor cells (EPCs) are primitive bone marrow (BM) cells that possess the capacity to mature into endothelial cells and play a role in neovascularization and vascular remodelling. This study aimed to investigate whether KMUP-1, a synthetic xanthine-based derivative, atorvastatin and simvastatin, can prevent endothelial dysfunction and apoptosis induced by hypoxia and to elucidate the underlying mechanisms., Methods: Mononuclear cells were separated and were induced to differentiate into EPCs. KMUP-1, atorvastatin or simvastatin were administered prior to hypoxia., Key Findings: We found that EPCs exposed to hypoxia increased apoptosis as well as diminished proliferation. Pretreatment with KMUP-1, atorvastatin and simvastatin significantly prevented hypoxia-induced EPCs death and apoptosis, with associated increased of the Bcl-2/Bax ratio, and reduced caspase-3 and caspase-9 expression. We also assessed the nitrite production and Ser(1177)-phospho-eNOS expression and found that KMUP-1, atorvastatin and simvastatin not only increased the secretion of NO compared with the hypoxia group but also upregulated the eNOS activation., Conclusions: KMUP-1 inhibited hypoxia-induced dysfunction and apoptosis in EPCs, which may be mediated through suppressing oxidative stress, upregulating eNOS and downregulating the caspase-3 signalling pathway., (© 2016 Royal Pharmaceutical Society.)

KMUP-3 (7-{2-[4-(4-nitrobenzene) piperazinyl]ethyl}-1, 3-dimethylxanthine) displays cardioprotection and increases cardiac output, and is suggested to increase cardiac performance and improve myocardial infarction. To determine whether KMUP-3 improves outcomes in hypoperfused myocardium by inducing Ca(2+) sensitization to oppose protein kinase (PK)G-mediated Ca(2+) blockade, we measured left ventricular systolic blood pressure, maximal rates of pressure development, mean arterial pressure and heart rate in rats, and measured contractility and expression of PKs/RhoA/Rho kinase (ROCK)II in beating guinea pig left atria. Hemodynamic changes induced by KMUP-3 (0.5-3.0 mg/kg, intravenously) were inhibited by Y27632 [(R)-(+)-trans-4-1-aminoethyl)-N-(4-Pyridyl) cyclohexane carboxamide] and ketanserin (1 mg/kg, intravenously). In electrically stimulated left guinea pig atria, positive inotropy induced by KMUP-3 (0.1-100μM) was inhibited by the endothelial NO synthase (eNOS) inhibitors N-nitro-l-arginine methyl ester (L-NAME) and 7-nitroindazole, cyclic AMP antagonist SQ22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine], soluble guanylyl cyclase (sGC) antagonist ODQ (1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one), RhoA inhibitor C3 exoenzyme, β-blocker propranolol, 5-hydroxytryptamine 2A antagonist ketanserin, ROCK inhibitor Y27632 and KMUP-1 (7-{2-[4-(2-chlorobenzene) piperazinyl]ethyl}-1, 3-dimethylxanthine) at 10μM. Western blotting assays indicated that KMUP-3 (0.1-10μM) increased PKA, RhoA/ROCKII, and PKC translocation and CIP-17 (an endogenous 17-kDa inhibitory protein) activation. In spontaneous right atria, KMUP-3 induced negative chronotropy that was blunted by 7-nitroindazole and atropine. In neonatal myocytes, L-NAME inhibited KMUP-3-induced eNOS phosphorylation and RhoA/ROCK activation. In H9c2 cells, Y-27632 (50μM) and PKG antagonist KT5823 [2,3,9,10,11,12-hexahydro-10R- methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl) pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid, methyl ester] (3μM) reversed KMUP-3 (1-100μM)-induced Ca(2+)-entry blockade. GPCR agonist activity of KMUP-3 appeared opposed to KMUP-1, and increased cardiac output via Ca(2+) sensitization, and displayed cardioprotection via cyclic GMP/PKG-mediated myocardial preconditioning in animal studies., (Copyright © 2016. Published by Elsevier Taiwan.)

The phosphodiesterase inhibitor (PDEI)/eNOS enhancer KMUP-1, targeting G-protein coupled receptors (GPCRs), improves dyslipidemia. We compared its lipid-lowering effects with simvastatin and explored hormone-sensitive lipase (HSL) translocation in hepatic fat loss. KMUP-1 HCl (1, 2.5, and 5 mg/kg/day) and simvastatin (5 mg/kg/day) were administered in C57BL/6J male mice fed a high-fat diet (HFD) by gavage for 8 weeks. KMUP-1 inhibited HFD-induced plasma/liver TG, total cholesterol, and LDL; increased HDL/3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)/Rho kinase II (ROCK II)/PPARγ/ABCA1; and decreased liver and body weight. KMUP-1 HCl in drinking water (2.5 mg/200 ml tap water) for 1-14 or 8-14 weeks decreased HFD-induced liver and body weight and scavenger receptor class B type I expression and increased protein kinase A (PKA)/PKG/LDLRs/HSL expression and immunoreactivity. In HepG2 cells incubated with serum or exogenous mevalonate, KMUP-1 (10(-7)∼10(-5) M) reversed HMGR expression by feedback regulation, colocalized expression of ABCA1/apolipoprotein A-I/LXRα/PPARγ, and reduced exogenous geranylgeranyl pyrophosphate/farnesyl pyrophosphate (FPP)-induced RhoA/ROCK II expression. A guanosine 3',5'-cyclic monophosphate (cGMP) antagonist reversed KMUP-1-induced ROCK II reduction, indicating cGMP/eNOS involvement. KMUP-1 inceased PKG and LDLRs surrounded by LDL and restored oxidized LDL-induced PKA expresion. Unlike simvastatin, KMUP-1 could not inhibit (14)C mevalonate formation. KMUP-1 could, but simvastatin could not, decrease ROCK II expression by exogenous FPP/CGPP. KMUP-1 improves HDL via PPARγ/LXRα/ABCA1/Apo-I expression and increases LDLRs/PKA/PKG/HSL expression and immunoreactivity, leading to TG hydrolysis to lower hepatic fat and body weight., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Phosphodiesterase (PDE) inhibitors have been suggested as a possible candidate for the treatment of osteopenia, including osteoporosis. KMUP-1 is a novel xanthine derivative with inhibitory activities on the PDE 3, 4, and 5 iso-enzymes to suppress the degradation of cAMP and cGMP. This study aimed to investigate the effect of KMUP-1 on osteoblast differentiation and the underlying cellular and molecular mechanisms. Primary osteoblasts and osteoblastic MC3T3-E1 cells were examined. KMUP-1 enhanced alkaline phosphatase (ALP) activity and mineralization compared to untreated controls in primary osteoblasts and MC3T3-E1 cells. KMUP-1 also increased the mRNA expression of the osteoblastic differentiation markers, including collagen type 1a, ALP, osteocalcin, osteoprotegerin, BMP-2, and Runx2, a key transcription regulator for osteoblastic differentiation. The osteogenic effect of KMUP-1 was abolished by BMP signaling inhibitor, noggin. Furthermore, we found that KMUP-1 upregulated Smad1/5/8 phosphorylations with subsequent BRE-Luc activation confirmed by transient transfection assay. In addition, KMUP-1 inactivated glycogen synthase kinase-3β (GSK-3β), with associated nuclear translocation of β-catenin. Co-treatment with H89 and KT5823, cAMP and cGMP pathway inhibitors, respectively, reversed the KMUP-1-induced activations of Smad1/5/8, β-catenin, and Runx2. The findings demonstrate for the first time that KMUP-1 can promote osteoblast maturation and differentiation in vitro via BMP-2/Smad1/5/8 and Wnt/β-catenin pathways. These effects are mediated, in part, by the cAMP and cGMP signaling. Thus, KMUP-1 may be a novel osteoblast activator and a potential new therapy for osteoporosis., (© 2015 Wiley Periodicals, Inc.)

The signaling cascades of the mitogen activated protein kinase (MAPK) family, calcineurin/NFATc4, and PI3K/Akt/GSK3, are believed to participate in endothelin-1 (ET-1)-induced cardiac hypertrophy. The aim of this study was to investigate whether KMUP-1, a synthetic xanthine-based derivative, prevents cardiomyocyte hypertrophy induced by ET-1 and to elucidate the underlying mechanisms. We found that in H9c2 cardiomyocytes, stimulation with ET-1 (100 nM) for 4 days induced cell hypertrophy and enhanced expressions of hypertrophic markers, including atrial natriuretic peptide and brain natriuretic peptide, which were all inhibited by KMUP-1 in a dose-dependent manner. In addition, KMUP-1 prevented ET-1-induced intracellular reactive oxygen species generation determined by the DCFH-DA assay in cardiomyocytes. KMUP-1 also attenuated phosphorylation of ERK1/2 and Akt/GSK-3β, and activation of calcineurin/NFATc4 and RhoA/ROCK pathways induced by ET-1. Furthermore, we found that the expression of heme oxygenase-1 (HO-1), a stress-response enzyme implicated in cardio-protection, was up-regulated by KMUP-1. Finally, KMUP-1 attenuated ET-1-stimulated activator protein-1 DNA binding activity. In conclusion, KMUP-1 attenuates cardiomyocyte hypertrophy induced by ET-1 through inhibiting ERK1/2, calcineurin/NFATc4 and RhoA/ROCK pathways, with associated cardioprotective effects via HO-1 activation. Therefore, KMUP-1 may have a role in pharmacological therapy of cardiac hypertrophy.

Ascorbic acid bound to KMUP-1 and sildenafil were examined for their antioxidant effects on vascular endothelium growth factor (VEGF) and endothelium nitric oxide synthase (eNOS) in hypoxic pulmonary artery (PA). Inhaled KMUP-1 and oral sildenafil released NO from eNOS. The effect of buffered l-ascorbic acid, alone and bound to KMUP-1 or sildenafil, for treating pulmonary arterial hypertension (PAH) is unclear. In this study, the antioxidant capacity of ascorbic acid increased the beneficial effects of KMUP-1 on PAH. KMUP-1A and sildenafil-A (5 mg/kg/d) were administered to hypoxic PAH rats. Pulmonary artery blood pressure, and VEGF, Rho kinase II (ROCK II), eNOS, soluble guanylate cyclase (sGC-α), and protein kinase G expression in lung tissues were measured to link PAH and right ventricular hypertrophy. Hypoxic rats had higher pulmonary artery blood pressure, greater PA medial wall thickness and cardiac weight, and a higher right ventricle/left ventricle + septum [RV/(LV+S)] ratio than normoxic rats. Oral KMUP-1A or sildenafil-A for 21 days in hypoxia prevented the rarefaction of eNOS in immunohistochemistry (IHC), reduced the IHC of VEGF in PAs, restored eNOS/protein kinase G/phosphodiesterase 5A; unaffected sGC-α and inactivated ROCK II expression were also found in lung tissues. In normoxic PA, KMUP-1A/Y27632 (10μM) increased eNOS and reduced ROCK II. ROCK II/reactive oxidative species was increased and eNOS was reduced after long-term hypoxia for 21 days. KMUP-1A or Y27632 blunted ROCK II in short-term hypoxic PA at 24 hours. l-Ascorbic acid + l-sodium ascorbate (40, 80μM) buffer alone directly inhibited the IHC of VEGF in hypoxic PA. Finally, KMUP-1A or sildenafil-A reduced PAH and associated right ventricular hypertrophy., (Copyright © 2015. Published by Elsevier Taiwan.)

Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor that promotes pulmonary artery smooth muscle cell (PASMC) proliferation. 5-HT-induced K(+) channel inhibition increases [Ca(2+)]i in PASMCs, which is a major trigger for pulmonary vasoconstriction and development of pulmonary arterial hypertension (PAH). This study investigated whether KMUP-1 reduces pulmonary vasoconstriction in isolated pulmonary arteries (PAs) and attenuates 5-HT-inhibited K(+) channel activities in PASMCs. In endothelium-denuded PA rings, KMUP-1 (1 μM) dose-dependently reduced 5-HT (100 μM) mediated contractile responses. Responses to KMUP-1 were reversed by K(+) channel inhibitors (TEA, 10 mM, 4-aminopyridine, 5 mM, and paxilline, 10 μM). In primary PASMCs, KMUP-1 also dose-dependently restored 5-HT-inhibited voltage-gated K(+)-channel (Kv1.5 and Kv2.1) and large-conductance Ca(2+)-activated K(+)-channel (BKCa) proteins, as confirmed by immunofluorescent staining. Furthermore, 5-HT (10 μM)-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 (1 μM) and the PKC activator PMA (1 μM), but these effects were reversed by KMUP-1 (1 μM), 8-Br-cAMP (100 μM), chelerythrine (1 μM), and KMUP-1 combined with a PKA/PKC activator or inhibitor. Notably, KMUP-1 reversed 5-HT-inhibited Kv1.5 protein and this response was significantly attenuated by co-incubation with the PKC activator PMA, suggesting that 5-HT-mediated PKC signaling can be modulated by KMUP-1. In conclusion, KMUP-1 ameliorates 5-HT-induced vasoconstriction and K(+)-channel inhibition through the PKC pathway, which could be valuable to prevent the development of PAH.

This study investigates whether endothelin-1 (ET-1) mediates monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and right ventricular hypertrophy (RVH), and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl}-1,3-dimethylxanthine) inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg) induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day) for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS) immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII) in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg) maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 μM), KMUP-1 (0.1-100 μM) inhibited ET-1 (0.01 μM)-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs), KMUP-1 (0.1-10 μM) inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in the treatment of PAH caused by MCT., (Copyright © 2014. Published by Elsevier B.V.)

Neuropathic pain is characterized by spontaneous pain, hyperalgesia, and allodynia. The aim of this study was to investigate whether KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) could improve pain hypersensitivity and reduce inflammatory mediators, and also explore possible mechanisms in the rat sciatic nerve using bilateral chronic constriction injury (CCI) to induce neuropathic pain. Sprague-Dawley rats were randomly divided into four groups: Sham, Sham+KMUP-1, CCI, and CCI+KMUP-1. KMUP-1 (5 mg/kg/day) was injected intraperitoneally starting at day 1 after surgery. Mechanical and thermal responses were assessed before surgery and at days 3, 7, and 14 after CCI. Sciatic nerves around the injury site were isolated for Western blots and enzyme-linked immunosorbent assay to analyze protein and cytokine levels. The results show that thermal hyperalgesia and mechanical allodynia were reduced in the KMUP-1 treated group as compared to that in the CCI group. Inflammatory proteins (COX2, iNOS, and nNOS) and proinflammatory cytokines (TNF-α and IL-1β) induced by CCI were decreased in the KMUP-1 treated group at day 7 after surgery. KMUP-1 also inhibited neuropathic pain-related mechanisms, including p38 and ERK activation, but not JNK. Furthermore, KMUP-1 blocked IκB phosphorylation (p-IκB) and phospho-nuclear factor κB (p-NF-κB) translocation to nuclei. Double immunofluorescent staining further demonstrated that p-IκB (an indicator of activated NFκB) and p-NFκB proteins were almost abolished by KMUP-1 in peripheral macrophages and spinal microglia cells at day 7 after surgery. On the basis of these findings, we concluded that KMUP-1 has antiinflammatory and antihyperalgesia properties in CCI-induced neuropathic pain via decreases in MAPKs and NF-κB activation.

Objectives: To investigate whether 7-[2-[4-(2-chlorophenyl) piperazinyl] ethyl]-1,3-di-methylxanthine (KMUP-1) inhibits the effects of testosterone on the development of benign prostatic hyperplasia and sensitizes prostate contraction., Methods: A benign prostatic hyperplasia animal model was established by subcutaneous injections of testosterone (3 mg/kg/day, s.c.) for 4 weeks in adult male Sprague-Dawley rats. Animals were divided into six groups: control, testosterone, testosterone with KMUP-1 (2.5, 5 mg/kg/day), sildenafil (5 mg/kg/day) or doxazosin (5 mg/kg/day). After 4 weeks, the animals were killed, and prostate tissues were prepared for isometric tension measurement and western blotting analysis. KMUP-1, Y27632, zaprinast, doxazosin or tamsulosin were used at various concentrations to determine the contractility sensitized by phenylephrine (10 μmol/L)., Results: KMUP-1 inhibited testosterone-induced phosphorylation of extracellular signal-regulated phosphorylated protein kinase and mitogen-activated protein kinase kinase and Rho kinase-II activation. Sildenafil and doxazosin significantly decreased benign prostatic hyperplasia-induced mitogen-activated protein kinase kinase and Rho kinase-II activation. The decreased expressions of soluble guanylate cyclase α1 was reversed by KMUP-1, doxazosin and sildenafil. Soluble guanylate cyclase β1 and protein kinase G were increased by KMUP-1, doxazosin, and sildenafil in the testosterone-treated benign prostatic hyperplasia group. Phosphodiesterase-5A was increased by testosterone and inhibited by KMUP-1 (5 mg/kg/day) or sildenafil (5 mg/kg/day). KMUP-1 inhibited phenylephrine-sensitized prostate contraction of rats treated with testosterone., Conclusions: Mitogen-activated protein kinase 1/extracellular regulated protein kinases kinase, soluble guanylate cyclase/cyclic guanosine monophosphate, protein kinase/protein kinase G and Rho kinase-II are related to prostate smooth muscle tone and proliferation induced by testosterone. KMUP-1 inhibits testosterone-induced prostate hyper-contractility and mitogen-activated protein kinase 1/extracellular regulated protein kinases kinase-phosphorylation, and it inactivates Rho kinase-II by cyclic guanosine monophosphate, protein kinase and α1A-adenergic blockade. Thus, KMUP-1 might be a beneficial pharmacotherapy for benign prostatic hyperplasia., (© 2013 The Japanese Urological Association.)

Background: KMUP-1 is a xanthine derivative with inhibitory activities on the phosphodiesterase (PDE) 3,4 and 5 isoenzymes to suppress the degradation of cyclic AMP and cyclic GMP. However, the effects of KMUP-1 on osteoclast differentiation are still unclear. In this study, we investigated whether KMUP-1 inhibits osteoclastogenesis induced by RANKL in RAW 264.7 cells and bone loss induced by ovariectomy in mice, and the underlying mechanisms., Principal Findings: In vitro, KMUP-1 inhibited RANKL-induced TRAP activity, the formation of multinucleated osteoclasts and resorption-pit formation. It also inhibited key mediators of osteoclastogenesis including IL-1β, IL-6, TNF-α and HMGB1. In addition, KMUP-1 inhibited RANKL-induced activation of signaling molecules (Akt, MAPKs, calcium and NF-κB), mRNA expression of osteoclastogensis-associated genes (TRAP, MMP-9, Fra-1, and cathepsin K) and transcription factors (c-Fos and NFATc1). Furthermore, most inhibitory effects of KMUP-1 on RANKL-mediated signal activations were reversed by a protein kinase A inhibitor (H89) and a protein kinase G inhibitor (KT5823). In vivo, KMUP-1 prevented loss of bone mineral content, preserved serum alkaline phosphate and reduced serum osteocalcin in ovariectomized mice., Conclusions: KMUP-1 inhibits RANKL-induced osteoclastogenesis in vitro and protects against ovariectomy-induced bone loss in vivo. These effects are mediated, at least in part, by cAMP and cGMP pathways. Therefore, KMUP-1 may have a role in pharmacologic therapy of osteoporosis.

Delayed cerebral vasospasm is a main cause of morbidity and mortality as well as poor outcome in patients following aneurysmal subarachnoid hemorrhage (SAH). In this study, the effect of the bronchodilator KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) on basilar artery narrowing, neurological outcome, and expression of rhoA/rho kinase II (ROCKII), rhoA, and protein kinase C (PKC) γ proteins were evaluated in a rat model of SAH. SAH was induced by double injection of autologous blood into the cistern magna on days 0 and 3. KMUP-3 was administered (0.3 mg/kg/day) by osmotic minipumps implanted subcutaneously (beginning day -3 in pretreatment group and at 1 h after the initiation of the first autologous blood injection in the treatment group). Neurological outcome was assessed by ambulation and placing/stepping reflex responses at 48 h after the second injection of autologous blood. Tissue morphology and protein expression were conducted on day 7 post-day 0 injection. Both KMUP-3 treatment regimens significantly improved neurological outcome and completely attenuated basilar artery narrowing as well as reduced the enhancement of ROCKII, rhoA, and PKCγ protein expression in rats subjected to SAH, compared with normal and untreated SAH rats. These results suggest that KMUP-3 may be a novel agent for the treatment of cerebral vasospasm following SAH.

Hypertension can induce left ventricular hypertrophy (LVH), and the nitric oxide (NO) pathway plays an important role in the pathogenesis of cardiac hypertrophy. This study aimed to examine whether KMUP-1, a novel xanthine-based derivative, could inhibit LVH in spontaneously hypertensive rats (SHRs) and to investigate potential mechanisms underlying its antihypertrophic effects. Two groups of animals with chronic or subacute LVH were treated. In the chronic LVH group, KMUP-1 (10 or 30 mg/kg/d orally) was administered for 28 days to both normotensive rats and SHRs. In the subacute LVH group, KMUP-1 (0.5 mg/kg/d intraperitoneally) or sildenafil (0.7 mg/kg/d intraperitoneally) was administered for 10 days with or without co-treatment with the nitric oxide synthase (NOS) inhibitor N-omega-nitro-l-arginine (L-NNA; 20 mg/L orally). After treatment, the effects of KMUP-1 or sildenafil on hypertension, cardiac hypertrophy, survival, expression of the NO/soluble guanylate cyclase (sGC)/protein kinase G (NO/sGC/PKG) pathway in the aorta andleft ventricle, and calcineurin A/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the left ventricle were examined. In the chronic LVH group, the SHRs developed hypertension with LVH over the 28 days. KMUP-1 attenuated the hypertension and LVH, increased survival rate, enhanced endothelial NOS/cyclic guanosine monophosphate/PKG (eNOS/cGMP/PKG) and decreased inducible NOS (iNOS) expression in the aorta and left ventricle of the SHRs. In the subacute LVH group, both KMUP-1 and sildenafil administered for 10 days attenuated the LVH in SHRs, with enhanced eNOS/cGMP/PKG and suppressed iNOS/calcineurin A/ERK1/2 expression in the left ventricle. In addition, both KMUP-1 and sildenafil attenuated L-NNA-induced LVH. KMUP-1 inhibition of hypertension-induced LVH with associated upregulation of eNOS, downregulation of iNOS in both the aorta and left ventricle, and attenuation of calcineurin A and ERK1/2 signaling in the left ventricle., (Copyright © 2012. Published by Elsevier B.V.)

Alterations in the activity of vascular K channels are commonly associated with abnormalities in cerebral vascular function after subarachnoid hemorrhage (SAH). Subarachnoid hemorrhage-induced vasospasm remains incompletely understood; nevertheless, activation of K channels may be of benefit in relieving spastic constriction. This study was to examine whether the vasodilators KMUP-1 and pinacidil, a KATP-channel opener, have the ability to prevent SAH-induced vasospasm via the large-conductance Ca-activated K (BKCa) channels in cerebral arteries. Rats were divided into four groups including sham-operated, SAH, KMUP-1 treated, and pinacidil treated. Subarachnoid hemorrhage rats were induced by injecting autologous blood into cisterna magna, and then KMUP-1 or pinacidil (1 mg/kg) was injected intraperitoneally 1 and 24 h after SAH. Behavioral tests were assessed on day 2 after SAH before the rats were killed. Cerebral myocytes were enzymatically isolated from rat basilar arteries and used to monitor BKCa-channel activities. In isolated basilar arteries, KMUP-1-treated and pinacidil-treated rats showed normalized vascular reactivity. In whole-cell recordings, BKCa currents were attenuated in SAH rats compared with sham-operated rats. In inside-out patches, the conductance and voltage sensitivity of single BKCa-channels were unchanged among the four groups. In contrast, SAH rats showed markedly decreased BKCa-channel activity and β1-subunit expression associated Ca sensitivity that was further confirmed by immunofluorescence staining and Western blotting. Subarachnoid hemorrhage-induced deficits in motor function and BKCa-channel inhibition were improved by KMUP-1-treated and pinacidil-treated rats. In addition, SAH appears to modify BKCa-channel calcium sensitivity. KMUP-1 and pinacidil prevent SAH-induced vasospasm at least in part by the restoration of BKCa-channel activities.

Aims: This study determines whether KMUP-1 inhalation suppresses ovalbumine (OVA)-sensitized and - challenged peri-bronchial vascular inflammation and remodeling in mice., Methods and Results: After short-term KMUP-1 (1-5 mM, 30 min)-nebulization and L-NAME (12 mM, 15 min)- pretreatment, endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases-9 (MMP-9) expression in lung were measured by Western blotting analysis. In 28-days experiment, mice were sensitized with intraperitoneal OVA on day 1 and day 8, challenged with OVA nebulization and treated with KMUP-1 nebulization (5 mM, 30 mins) on day 21-27. Expression of eNOS, inducible nitric oxide synthase (iNOS), soluble guanylyl cyclase (sGC), protein kinase G (PKG), MMP-9, VCAM-1 and ICAM-1 were measured by Western blotting analysis. eNOS- and MMP-9-immunostaining were used for peri-vascular or peri-bronchial localization. Hematoxylin and eosin staining was used to show the vascular and bronchial wall thickness and infiltration of inflammatory cells. Cell counting and measurement of NOmetabolite (NOx) in bronchoalveolar lavage fluid (BALF) were used to examine the NO production. KMUP-1 increased eNOS and decreased MMP-9 expression. L-NAME-pretreatment reversed these changes. KMUP-1 reduced OVA-sensitized vascular and bronchial wall thickening, eNOS-immunostaining at the alveolar septa, MMP-9-immunostaining in the bronchioles and infiltrated inflammatory cells in the peri-vascular and peri-bronchiolar regions. The OVA-sensitized decrease of sGC and PKG and increase of iNOS, ICAM-1/VCAM-1 and plasma cytokines IL-5/IL-13 were reversed; cell count, NOx and MMP-9-activity in BALF were decreased by KMUP-1., Conclusions: Inhaled KMUP-1, preventing allergic pulmonary vascular inflammation and remodeling, would be useful for the treatment of asthma and respiratory obstruction disease.

This study investigated whether KMUP-1, a xanthine-based derivative, inhibits L-type Ca(2+) currents (I(Ca,L)) in rat basilar artery smooth muscle cells (RBASMCs). We used whole cell patch-clamp recording to monitor Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels (LTCCs). Under voltage-clamp conditions, holding at -40 mV, KMUP-1 (1, 3, 10 μM) inhibited I(Ba) in a concentration-dependent manner and its IC(50) value was 2.27 ± 0.45 μM. A high concentration of KMUP-1 (10 μM) showed without modifying the I(Ba) current-voltage relationship. On the other hand, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM) increase I(Ba) was inhibited by KMUP-1. Pretreatment with the PKC inhibitor chelerythrine (5 μM) intensified KMUP-1-inhibited I(Ba). However, the Rho kinase inhibitor Y-27632 (30 μM) failed to affect the I(Ba) inhibition by KMUP-1. In light of these results, we suggest that KMUP-1 inhibition of LTCCs in concentration- and voltage-dependent manners in RBASMCs may be due, at least in part, to its modulation of the PKC pathway., (Copyright © 2011 Elsevier Taiwan LLC. All rights reserved.)

Background and Purpose: Previously, 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) has been shown to induce aortic smooth muscle relaxation through K(ATP) channel opening and endothelial nitric oxide synthase (eNOS) enhancement. We further investigated whether KMUP-3 protects against myocardial remodelling after myocardial infarction (MI), and whether KMUP-3 increases the expression of eNOS in MI rats., Experimental Approach: Wistar rats were randomly allocated into three groups: MI (n= 10), MI + KMUP-3 group (n= 10) and sham group (n= 10). MI was induced by ligation of the left anterior descending coronary artery. After recovery, the MI + KMUP-3 group received KMUP-3 (0.3 mg·kg(-1) ·day(-1) ) infusion for 4 weeks, while the MI and sham group received vehicle only. To further confirm that the effect of KMUP-3 is dependent on eNOS, KMUP-3 was applied in the culture of transforming growth factor-β-stimulated human cardiac fibroblasts., Key Results: KMUP-3 treatment attenuated cardiac hypertrophy post-MI and improved cardiac function. The fibrotic area was reduced by KMUP-3 both in central-, peri- and non-infarction areas. KMUP-3 enhanced the expression of eNOS and tissue inhibitor of metalloproteinase-1 (TIMP-1), but reduced matrix metalloproteinase-9 (MMP-9) expression. In vitro, the activities of KMUP-3 were blocked by pretreatment with the eNOS inhibitor N(ω) -nitro-L-arginine methyl ester., Conclusions and Implications: The K(ATP) channel opener KMUP-3 preserved cardiac function after MI by enhancing the expression of eNOS. In addition, KMUP-3 restored the myocardial MMP-9/TIMP-1 balance and attenuated ventricular remodelling by an eNOS-dependent mechanism., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background and Purpose: To determine whether KMUP-1, a novel xanthine-based derivative, attenuates isoprenaline (ISO)-induced cardiac hypertrophy in rats, and if so, whether the anti-hypertrophic effect is mediated by the nitric oxide (NO) pathway., Experimental Approach: In vivo, cardiac hypertrophy was induced by injection of ISO (5 mg.kg(-1).day(-1), s.c.) for 10 days in Wistar rats. In the treatment group, KMUP-1 was administered 1 h before ISO. After 10 days, effects of KMUP-1 on survival, cardiac hypertrophy and fibrosis, the NO/guanosine 3'5'-cyclic monophosphate (cGMP)/protein kinase G (PKG) and hypertrophy signalling pathways [calcineurin A and extracellular signal-regulated kinase (ERK)1/2] were examined. To investigate the role of nitric oxide synthase (NOS) in the effects of KMUP-1, a NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA) was co-administered with KMUP-1. In vitro, anti-hypertrophic effects of KMUP-1 were studied in ISO-induced hypertrophic neonatal rat cardiomyocytes., Key Results: In vivo, KMUP-1 pretreatment attenuated the cardiac hypertrophy and fibrosis and improved the survival of ISO-treated rats. Plasma NOx (nitrite and nitrate) and cardiac endothelial NOS, cGMP and PKG were all increased by KMUP-1. The activation of hypertrophic signalling by calcineurin A and ERK1/2 in ISO-treated rats was also attenuated by KMUP-1. All these effects of KMUP-1 were inhibited by simultaneous administration of L-NNA. Similarly, in vitro, KMUP-1 attenuated hypertrophic responses and signalling induced by ISO in neonatal rat cardiomyocytes., Conclusions and Implications: KMUP-1 attenuates the cardiac hypertrophy in rats induced by administration of ISO. These effects are mediated, at least in part, by NOS activation. This novel agent, which targets the NO/cGMP pathway, has a potential role in the prevention of cardiac hypertrophy.

The present study was performed to elucidate the effects of itraconazole and rifampin on the pharmacokinetics and pharmacodynamics of ebastine, a nonsedative H1 receptor antagonist. In a 3-way crossover sequential design with 2-week washouts, 10 healthy participants were pretreated with itraconazole for 6 days, rifampin for 10 days, or neither. After oral administration of 20 mg ebastine, blood and urine samples were collected for 72 and 24 hours, respectively, and histamine-induced wheal and flare reactions were measured to assess the antihistamine response for 12 hours. Itraconazole pretreatment decreased the oral clearance of ebastine to 10% (P < .001) and increased the AUC(infinity) of the active metabolite, carebastine, by 3-fold (P < .001). On the other hand, rifampin pretreatment decreased the AUC(infinity) of carebastine to 15% (P < .001), with an enormous reduction in the oral bioavailability of ebastine and significantly reduced histamine-induced skin reactions. From these results, the disposition of ebastine and carebastine seems to be significantly altered by coadministration of itraconazole or rifampin. The antihistamine response after ebastine dosing would be decreased following rifampin pretreatments.

Aging populations with neurodegenerative disorders will gradually become a greater problem for society. Serum deprivation-induced cell death is recognized as one of the standard models for the study of neurotoxicity. Increasing evidence indicates that cGMP/PKG pathway may play a rescue role in serum deprivation-induced toxicity. The aim of this study was to investigate protective effects of KMUP-1, an enhancer of cGMP/PKG signaling on serum deprivation-induced neurotoxicity in SH-SY5Y neuroblastoma cells. Under normal serum condition, KMUP-1 enhanced protein expression of nNOS, PKG and sGCalpha1, increased intracellular cyclic GMP level, and attenuated PDE5 expression. KMUP-1 also increased expression of BDNF and Bcl-2, but it did not affect Bax expression. The phosphorylation of Akt and CREB induced by KMUP-1 was inhibited by tyrosine kinase (TrK) inhibitor K252a and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, respectively. Under serum deprivation condition, flow cytometric analysis using Annexin V showed KMUP-1 increased cell viability, but lacked protective effects in the presence of nitric oxide synthase inhibitor l-NAME, PKG inhibitor Rp-8-pCPT-cGMPS or LY294002. KMUP-1 not only enhanced expression of nNOS, sGCalpha1, PKG, p-CREB, p-Akt and Bcl-2, but also attenuated Bax expression in serum deprivation-treated cultures. In conclusion, cGMP/PKG, PI3K/Akt/CREB and Bcl-2/Bax signals play critical roles in the neuroprotective effects of KMUP-1 on serum deprivation-induced toxicity., (2009 Elsevier Ireland Ltd. All rights reserved.)

Background: KMUP-1 has been suggested to be beneficial in the treatment of benign prostatic hyperplasia. This study is aimed to further investigate whether KMUP-1 and doxazosin prevent from prostate cancer cell growth via androgen-dependent and -independent pathway in vivo and in vitro., Methods: KMUP-1 was measured the activity on proliferation, apoptosis and cell cycle distribution in prostate cancer cells (LNCaP, DU-145, PC-3) by MTT assay, flow cytometry, Western Blotting and enzyme-linked immunosorbent assay (ELISA). The inhibition activities on androgen receptor (AR) and AR-targeting molecular prostate-specific antigen (PSA) expression by KMUP-1 and doxazosin were measured by RT-PCR, Western Blotting, and ELISA. Furthermore, we confirmed the effects of KMUP-1 on growth of LNCaP xenografts in nude mice., Results: KMUP-1 significantly inhibited LNCaP cell growth and induced apoptosis in time- and dose-dependent manner. KMUP-1 and doxazosin further inhibited the expression of AR and PSA. Treatment of LNCaP cells with KMUP-1 resulted in cell cycle arrest and apoptotic activities, increasing p21 and p27 and decreasing expressions of cyclin D1, cyclin E, cyclin dependent kinase (CDK) 4, CDK2 and CDK6. Moreover, KMUP-1 activated p53, cleaved poly (ADP-ribose) polymerase and caspase-3, but reduced the expression of Bcl-2. Regular administration of KMUP-1 suppressed the LNCaP xenograft tumor growth in nude mice., Conclusion: These evidences indicate that KMUP-1 and doxazosin inhibit LNCaP cell growth and downregulate expression of AR and PSA. KMUP-1 might be used as a chemoprevention agent for preventing the development of prostate cancer without cardiovascular adverse effect of doxazosin., ((c) 2009 Wiley-Liss, Inc.)

Background: Soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) and Rho kinase (ROCK2) pathways are important in the regulation of prostate smooth muscle tone. This study is aimed to examine the relaxation activities of a sGC activator and PDE5A/ROCK2 inhibitor KMUP-1 in rat prostate and associated anti-proliferation activity in human prostatic epithelial cells., Methods: The action characteristics of KMUP-1 were identified by isometric tension measurement, receptor binding assay, Western blotting and radioimmunoassay in rat prostate. Anti-proliferation activity of KMUP-1 in human prostatic epithelial PZ-HPV-7 cells was identified using flow cytometry and real time QRT-PCR., Results: KMUP-1 inhibited phenylephrine-induced contractility in a concentration-dependent manner. KMUP-1 possessed potent alpha(1A/)alpha(1D)-adrenoceptor binding inhibition activity, increased cAMP/cGMP levels and increased the expression of sGC, PKG, and PKA protein in rat prostate. Moreover, KMUP-1 inhibited phenylephrine-induced ROCK2 expression. KMUP-1 inhibited cell growth, arrested the cell cycle at G(0)/G(1) phase and increased the expression of p21 in PZ-HPV-7 cells., Conclusions: These results broaden our knowledge of sGC/cGMP/PKG and ROCK2 regulation on the relaxation and proliferation of prostate, which may help in the design of benign prostate hyperplasia (BPH) therapies that target these signaling pathways. KMUP-1 possesses the potential benefit in the treatment of BPH by its alpha(1A/)alpha(1D)-adrenoceptor blockade, sGC activation, inhibition of PDE5A and ROCK2 and p21 protein enhancement, leading to attenuation of the smooth muscle tone and the proliferation of epithelial PZ-HPV-7 cells. The synergistic contribution of these pathways by KMUP-1 may benefit BPH patients with lower urinary tract symptoms., (2007 Wiley-Liss, Inc)

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.

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 microl) induced bronchoconstriction, increases of lung resistance, and decreases of dynamic lung compliance. Instillation of KMUP-3 (0.5-2.0 mg/kg) reversed this situation. In isolated trachea precontracted with carbachol, KMUP-3 (10-100 microM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K(+) channel, tetraethylammonium (10 mm); K(ATP) channel, glibenclamide (1 microM); voltage-dependent K(+) channel, 4-aminopyridine (100 microM); Ca(2+)-dependent K(+) channel, charybdotoxin (0.1 microM) or apamin (1 microM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 microM); nitric-oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 microM). KMUP-3 (0.01-100 microM) induced increases of cGMP and cAMP in primary culture of tracheal smooth muscle cells (TSMCs). The increase in cGMP by KMUP-3 was reduced by ODQ and L-NAME; the increase in cAMP was reduced by SQ 22536. Western blot analysis indicated that KMUP-3 (1 microM) induced expression of protein kinase A (PKA)(ri) and protein kinase G (PKG)(1alpha 1beta) in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)(ri). On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1alpha 1beta) In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases in TSMCs by KMUP-3 may result in increases of intracellular cGMP and cAMP, which subsequently activate PKG and PKA, efflux of K(+) ion, and associated reduction in Ca(2+) influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.

The cellular mechanisms of vasorelaxant effects of newly synthesized KMUP-3 and KMUP-4 were investigated in rat aortic smooth muscle (RASM). KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) and KMUP-4 (7-[2-[4-(2-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) elicited concentration-dependent relaxation of endothelium-intact and denuded RASM precontracted with phenylephrine. Relaxant responses were also produced by the PDE inhibitors theophylline, milrinone, rolipram, and zaprinast (1 nM-100 microM). The relaxant responses of KMUP-3 and KMUP-4 were reduced by endothelium removal and by the presence of the NOS inhibitor L-NAME (100 microM), the sGC inhibitor ODQ (1 microM), the adenylyl cyclase (AC) inhibitor SQ 22536 (100 microM), and the prostaglandin inhibitor indomethacin (10 microM). Additionally, the vasorelaxations of both agents were also attenuated by pretreatment with the nonselective K+ channel blocker TEA (10 mM), the KATP channel blocker glibenclamide (1 microM), the voltage-dependent K+ (KV) channel blocker 4-AP (100 microM), and Ca(2+)-dependent K+ (KCa) channel blockers apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). In addition, elevated extracellular K+ (80 mM) interferes with KMUP-3- and KMUP-4-induced vasorelaxations. Preincubation with both agents (1 microM) significantly enhanced the dilator responses of isoproterenol and SNP. KMUP-3 and KMUP-4 inhibited PDE activities and increased cAMP and cGMP levels in primary culture of RASM that were inhibited by SQ 22536 and ODQ, respectively. In cultured HUVECs, KMUP-3 and KMUP-4 (0.1 microM), more potent than YC-1, significantly increased the expression of eNOS protein. In summary, KMUP-3 and KMUP-4 induce aortic relaxations through both endothelium-dependent and -independent mechanisms. Mechanisms of vasorelaxation induced by both compounds involve multiple processes, such as accumulation of cyclic nucleotides partly as a result of PDE inhibition, K-channel activation, and indomethacin-sensitive endothelium function.

This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (K(DR))- or large-conductance Ca2+-activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+- and Ca2+ channel activities. KMUP-1 (1 microM) had no effect on the K(DR) current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 microM), an adenylate cyclase inhibitor (SQ 22536 10 microM), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 microM and Rp-cAMP, 100 microM), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 microM), and nearly abolished by a calcium channel blocker (nifedipine, 1 microM). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries.

7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) produces tracheal relaxation, intracellular accumulation of cyclic nucleotides, inhibition of phosphodiesterases (PDEs) and activation of K+ channels. KMUP-1 (0.01-100 microm) induced concentration-dependent relaxation responses in guinea-pig epithelium-intact trachea precontracted with carbachol. Relaxation responses were also elicited by the PDE inhibitors theophylline, 3-isobutyl-1-methylxanthine (IBMX), milrinone, rolipram and zaprinast (100 microm), and a KATP channel opener, levcromakalim. Tracheal relaxation induced by KMUP-1 was attenuated by epithelium removal and by pretreatment with inhibitors of soluble guanylate cyclase (sGC) (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 1 microm), nitric oxide synthase (Nomega-nitro-L-arginine methyl ester, 100 microm), K+ channels (tetraethylammonium, 10 mm), KATP channels (glibenclamide, 1 microm), voltage-dependent K+ channels (4-aminopyridine, 100 microm) and Ca2+-dependent K+ channels (charybdotoxin, 0.1 microm or apamin, 1 microm). Both KMUP-1 (10 microm) and theophylline nonselectively and slightly inhibited the enzyme activity of PDE3, 4 and 5, suggesting that they are able to inhibit the metabolism of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP). Likewise, the effects of IBMX were also measured and its IC50 values for PDE3, 4 and 5 were 6.5 +/- 1.2, 26.3 +/- 3.9 and 31.7 +/- 5.3 microm, respectively. KMUP-1 (0.01-10 microm) augmented intracellular cyclic AMP and cyclic GMP levels in guinea-pig cultured tracheal smooth muscle cells. These increases in cyclic AMP and cyclic GMP were abolished in the presence of an adenylate cyclase inhibitor SQ 22536 (100 microm) and an sGC inhibitor ODQ (10 microm), respectively. KMUP-1 (10 microm) increased the expression of protein kinase A (PKARI) and protein kinase G (PKG1alpha1beta) in a time-dependent manner, but this was only significant for PKG after 9 h. Intratracheal administration of tumour necrosis factor-alpha (TNF-alpha, 0.01 mg kg(-1)) induced bronchoconstriction and exhibited a time-dependent increase in lung resistance (RL) and decrease in dynamic lung compliance (Cdyn). KMUP-1 (1.0 mg kg(-1)), injected intravenously for 10 min before the intratracheal TNF-alpha, reversed these changes in RL and Cdyn. These data indicate that KMUP-1 activates sGC, produces relaxation that was partly dependent on an intact epithelium, inhibits PDEs and increases intracellular cyclic AMP and cyclic GMP, which then increases PKA and PKG, leading to the opening of K+ channels and resulting tracheal relaxation., (Copyright 2004 Nature Publishing Group)

1. In isolated endothelium-intact or denuded rabbit corpus cavernosum preconstricted with phenylephrine, KMUP-1 (0.001 - 10 microM) caused a concentration-dependent relaxation. 2. This relaxation of KMUP-1 was attenuated by endothelium removed, high K(+) and pretreatments with a soluble guanylyl cyclase (sGC) inhibitor ODQ (1 microM), a NOS inhibitor L-NAME (100 microM), a K(+) channel blocker TEA (10 mM), a K(ATP) channel blocker glibenclamide (1 microM), a voltage-dependent K(+) channel blocker 4-AP (100 microM) and Ca(2+)-dependent K(+) channel blockers apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). 3. The relaxant responses of KMUP-1 (0.01, 0.05, 0.1 microM) together with a PDE inhibitor IBMX (0.5 microM) had additive actions on rabbit corpus cavernosum smooth muscle (CCSM). 4. KMUP-1 (0.01 - 10 microM) induced increase of intracellular cyclic GMP level in the primary cell culture of rabbit CCSM. This increase in cyclic GMP content was abolished in the presence of ODQ (10 microM). 5. Both KMUP-1 and sildenafil at 0.2, 0.4, 0.6 mg kg(-1) caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose-dependent manner. These in vivo activities of ICP for sildenafil and KMUP-1 are consistent with those of in vitro effects of cyclic GMP. 6. KMUP-1 has the following merits: (1) inhibition of PDE or cyclic GMP breakdown, (2) stimulation of NO/sGC/cyclic GMP pathway, and (3) subsequent stimulation of K(+) channels, in rabbit CCSM. We suggest that these merits play prominent roles in KMUP-1-induced CCSM relaxation-associated increases of ICP and penile erection.