piperidines and 7-(2-(4-(4-nitrobenzene)piperazinyl)ethyl)-1-3-dimethylxanthine

Inflammation, oxidative stress, matrix degradation, medial calcification and vascular smooth muscle cell (VSMC) loss are prominent features in abdominal aortic aneurysm (AAA). VSMC phenotypic switch to a proinflammatory state and VSMC apoptosis could be targetable mechanisms implicated in the pathogenesis of AAA formation. Herein, we investigated the hypothesis that a xanthine derivative (KMUP-3) might suppress AAA through inhibition of VSMC phenotypic switch and apoptosis.. In vitro, VSMC calcification was induced using β-glycerophosphate. In vivo, AAA was induced using angiotensin II (1000 ng/kg per minute) infusion for 4 weeks in apolipoprotein E-deficient mice.. As determined by alizarin red S staining and calcium content measurements, KMUP-3 suppressed VSMC calcification. During VSMC calcification, KMUP-3 inhibited mTOR and β-catenin upregulation, essential for VSMC phenotypic switch, while it enhanced AMP-activated protein kinase (AMPK) activation that protects against VSMC phenotypic switch. Moreover, KMUP-3 attenuated VSMC apoptosis with an increased Bcl-2/Bax ratio and reduced activated caspase-3 expression. During AAA formation, treatment with KMUP-3 inhibited phosphorylated mTOR expression and increased phosphorylated AMPK expression in the medial layer. In addition, KMUP-3 treatment suppressed aortic dilatation together with reduction in proinflammatory cytokines and infiltrating macrophages, attenuation of medial VSMC apoptosis and mitigation of reactive oxygen species generation, matrix-degrading proteinase activities, elastin breakdown and vascular calcification.. Treatment with KMUP-3 inhibits aneurysm growth possibly through its interference with signaling pathways involved in VSMC phenotypic switch and apoptosis. These findings provide a proof-of-concept validation for VSMC dysfunction as a potential therapeutic target in AAA.

Topics: Angiotensin II; Animals; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Apoptosis; Apoptosis Regulatory Proteins; Cells, Cultured; Disease Models, Animal; Male; Mice, Knockout, ApoE; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenotype; Piperidines; Rats, Sprague-Dawley; Signal Transduction; Vascular Calcification; Xanthines

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.

Topics: Animals; Blood Pressure; Calcium Signaling; Carbazoles; Cardiac Output; Cardiotonic Agents; Cell Line; Cyclic GMP-Dependent Protein Kinases; Drug Evaluation, Preclinical; Female; Guinea Pigs; Heart Atria; Male; Phosphodiesterase Inhibitors; Piperidines; Protein Transport; Rats, Wistar; Receptors, G-Protein-Coupled; Ventricular Pressure; Xanthines

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.

Topics: Animals; Bronchodilator Agents; Disease Models, Animal; Drug Interactions; Gene Expression Regulation; Hemodynamics; Locomotion; Male; Neurologic Examination; Piperidines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Reflex; rho-Associated Kinases; rhoA GTP-Binding Protein; Subarachnoid Hemorrhage; Vasospasm, Intracranial; Xanthines

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.. 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.. 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.. 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.

Topics: Animals; Blotting, Western; Cells, Cultured; Hemodynamics; Humans; Male; Matrix Metalloproteinase 9; Myocardial Infarction; Nitric Oxide Synthase Type III; Piperidines; Rats; Rats, Wistar; Tissue Inhibitor of Metalloproteinase-1; Ventricular Remodeling; Xanthines

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.

Topics: Adenylyl Cyclases; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Nitric Oxide; Phosphoric Diester Hydrolases; Piperidines; Potassium Channels; Respiration; Trachea; Tumor Necrosis Factor-alpha; Xanthines

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.

Topics: Animals; Cells, Cultured; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP-Dependent Protein Kinases; Cyclooxygenase 2; Guanylate Cyclase; Inflammation; Male; Models, Biological; Myocytes, Smooth Muscle; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrites; Nucleotides, Cyclic; Piperidines; Prostaglandins; Rats; Rats, Wistar; Solubility; Trachea; Tumor Necrosis Factor-alpha; Xanthines

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.

Topics: Animals; Aorta; Cell Line; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphoric Diester Hydrolases; Piperazines; Piperidines; Potassium Channels; Rats; Rats, Wistar; Vasoconstrictor Agents; Vasodilator Agents; Xanthines