Ferritinophagy plays a crucial role in ferroptosis, regulating cell death in response to reactive oxygen species (ROS) and iron metabolism. Geniposide (GEN) is an iridoid glycoside that has been reported to exhibit antioxidant and anti-inflammatory effects. Whether GEN can modulate hydrogen peroxide (H2O2)-induced inflammation and ferroptosis in human neuronal SH-SY5Y cells remains elusive. In the present study, we utilize the transwell co-culture system, which contains the human microglial cell line HMC3 (upper chamber) and the neuroblastoma cell line SH-SY5Y (lower chamber), to investigate the mechanisms of action of GEN and its neuroprotective effects. GEN inhibited ROS generation, decreased the malondialdehyde (MDA) level, attenuated p38 MAPK phosphorylation, nuclear receptor coactivator 4 (NCOA4), and SQSTM1 (p62) protein expression following H2O2 exposure. Meanwhile, GEN increases the intracellular GSH level and glutathione peroxidase 4 (GPX4) protein expression, protecting cells from oxidative damage. Therefore, GEN could modulate NCOA4-mediated ferritinophagic flux and alleviate ferroptosis. In addition, overexpression of NCOA4 significantly induced ferroptosis in both HMC3 and SH-SY5Y cells, but GEN mitigated these effects. NCOA4-mediated ferritinophagy collectively contributes to the process by which hydrogen peroxide induces oxidative stress and leads to cell ferroptosis. In conclusion, GEN alleviates hydrogen peroxide-induced inflammation and ferroptosis through the ROS/p38 MAPK/NCOA4/Gpx4-mediated ferritinophagy signaling pathway.

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in reduced dopamine levels in the striatum and eventual onset of motor symptoms. Linalool (3,7-dimethyl-1,6-octadien-3-ol) is a monoterpene in aromatic plants exhibiting antioxidant, antidepressant, and anti-anxiety properties. The objective of this study is to evaluate the neuroprotective impacts of linalool on dopaminergic SH-SY5Y cells, primary mesencephalic and cortical neurons treated with 1-methyl-4-phenylpyridinium ion (MPP+), as well as in PD-like mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Cell viability, α-tubulin staining, western blotting, immunohistochemistry and behavioral experiments were performed. In MPP+-treated SH-SY5Y cells, linalool increased cell viability, reduced neurite retraction, enhanced antioxidant defense by downregulation of apoptosis signaling (B-cell lymphoma 2 (Bcl-2), cleaved caspase-3 and poly ADP-ribose polymerase (PARP)) and phagocyte NADPH oxidase (gp91phox), as well as upregulation of neurotrophic signaling (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) and nuclear factor-erythroid 2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. In MPP+-treated primary mesencephalic neurons, linalool enhanced the expressions of tyrosine hydroxylase (TH), Sirtuin 1 (SirT1), and parkin. In MPP+-treated primary cortical neurons, linalool upregulated protein expression of SirT1, γ-Aminobutyric acid type A-α1 (GABAA-α1), and γ-Aminobutyric acid type B (GABAB). In PD-like mice, linalool attenuated the loss of dopamine neurons in SNpc. Linalool improved the motor and nonmotor behavioral deficits and muscle strength of PD-like mice. These findings suggest that linalool potentially protects dopaminergic neurons and improves the impairment symptoms of PD.

BACKGROUND:Methylglyoxal (MGO) is a potent precursor of glycative stress that leads to oxidative stress and muscle atrophy in diabetes. Spatheliachromen (FPATM-20), derived from Ficus pumila var. awkeotsang, exhibited potential antioxidant activity. PURPOSE:This study aimed to evaluate the potential impact and underlying mechanisms of FPATM-20 on MGO-induced myotube atrophy and mitochondrial dysfunction in mouse skeletal C2C12 myotubes. METHODS:Atrophic and antioxidant factors were evaluated using immunofluorescence, enzyme-linked immunosorbent assay, and western blotting. Mitochondrial function was assessed using the ATP assay and Seahorse Cell Mito Stress Test. The glycogen content was determined using periodic acid-Schiff staining. Molecular docking was performed to determine the interaction between FPATM-20 and Keap1. RESULTS:In myotubes treated with MGO, FPATM-20 activated the Nrf2 pathway, reduced ROS levels, enhanced antioxidant defense, and increased glycogen content. FPATM-20 improved myotube viability and size, upregulated myosin heavy chain (MyHC) expression, modulated ubiquitin-proteasome molecules (nuclear FoxO3a, atrogin-1, MuRF-1, and p62/SQSTM1), and inhibited apoptosis (Bax/Bcl-2 ratio and cleaved caspase 3). Moreover, FPATM-20 restored mitochondrial function, including mitochondrial membrane potential, mitochondrial oxygen consumption rate, and mitochondrial biogenesis pathway (nuclear PGC-1α/TFAM/FNDC5). The inhibition of Nrf2 with ML385 reversed the effects of FPATM-20 on MGO. Furthermore, molecular docking confirmed the binding of FPATM-20 to Keap1, a suppressor of Nrf2, showing the crucial role of Nrf2 in protective effects. CONCLUSIONS:FPATM-20 protects myotubes from MGO toxicity by activating the Nrf2 antioxidant defense, reducing protein degradation and apoptosis, and enhancing mitochondrial function. Thus, FPATM-20 may be a novel agent for preventing skeletal muscle atrophy.

Autophagy facilitates the degradation of organelles and cytoplasmic proteins in a lysosome-dependent manner. It also plays a crucial role in cell damage. Whether loganin affects autophagy in chronic constriction injury (CCI)-induced neuropathic pain remains unclear. We investigated the neuroprotective effect of loganin on the autophagic–lysosomal pathway in the rat CCI model. Sprague–Dawley rats were divided into sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was intraperitoneally injected once daily, and rats were sacrificed on day 7 after CCI. This study focused on the mechanism by which loganin modulates autophagic flux after CCI. CCI enhanced the autophagic marker LC3B-II in the ipsilateral spinal cord. The ubiquitin-binding protein p62 binds to LC3B-II and integrates into autophagosomes, which are degraded by autophagy. CCI caused the accumulation of p62, indicating the interruption of autophagosome turnover. Loganin significantly attenuated the expression of Beclin-1, LC3B-II, and p62. Double immunofluorescence staining was used to confirm that LC3B-II and p62 were reduced by loganin in the spinal microglia and astrocytes. Loganin also lessened the CCI-increased colocalization of both proteins. Enhanced lysosome-associated membrane protein 2 (LAMP2) and pro-cathepsin D (pro-CTSD) in CCI rats were also attenuated by loganin, suggesting that loganin improves impaired lysosomal function and autophagic flux. Loganin also attenuated the CCI-increased apoptosis protein Bax and cleaved caspase-3. Loganin prevents CCI-induced neuropathic pain, which could be attributed to the regulation of neuroinflammation, neuronal autophagy, and associated cell death. These data suggest autophagy could be a potential target for preventing neuropathic pain.

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.

This study examined the effects of eugenosedin-A (Eu-A) in a streptozotocin (STZ)/nicotinamide-induced rat model of type II diabetes mellitus (T2DM). Six-week-old Sprague-Dawley rats were randomly divided into three groups: (1) RD group, normal rats fed a regular diet (RD), (2) DM group, T2DM rats fed a high-fat diet, and (3) Eu-A group, T2DM rats fed a high fat diet plus oral Eu-A (5 mg/kg/day). After 30 days, the DM group had higher body weight, higher blood glucose and lower insulin levels than the RD group. The DM group also had increased protein expression of glycogen synthase kinase (GSK) in liver and skeletal muscle and decreased protein expression of insulin receptor (IR), insulin receptor substrate-1 (IRS1), IRS-2, AMP-activated protein kinase (AMPK), glucose transporter-4 (GLUT-4), glucokinase (GCK), and peroxisome proliferator-activated receptor gamma (PPAR-gamma). STZ/nicotinamide-induced T2DM increased the expression of mitogen-activated protein kinases (MAPKs: p38, ERK, JNK) and inflammatory p65 protein. In the Eu-A treated T2DM rats, however, blood glucose was attenuated and the insulin concentration stimulated. Changes in IR, IRS-1 and IRS-2 proteins as well as AMPK, GLUT-4, GCK, GSK, PPAR-gamma, MAPKs, and inflammatory p65 proteins were ameliorated. These results suggested that Eu-A alleviates STZ/nicotinamide-induced hyperglycemia by improving insulin levels and glucose metabolism, and inhibiting the MAPKs- and p65-mediated inflammatory pathway. Copyright (C) 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC.

Objectives Previous studies have shown eugenosedin-A, a 5-HT1B/2A and alpha(1)/alpha(2)/beta(1)-adrenergic blocker, is able to decrease cholesterol levels, hyperglycaemia and inflammation in hyperlipidaemic mice induced by high-fat diet (HFD). The aim of this study is to examine the effects of eugenosedin-A on the inhibition of adhesion molecules of platelets, the aorta and acyl-coenzymenA:cholesterol acyltransferase-1 (ACAT-1) of macrophages in a hyperlipidaemic rat model.Methods Six-week-old Sprague-Dawley rats were randomly divided into two control and treatment groups. The control rats received either a regular diet or HFD and the treatment groups were fed HFD with either 5 mg/kg eugenosedin-A or atorvastatin for a 10-week period.Key findings Compared with the two control groups, the HFD group had lower levels of high-density lipoprotein, higher concentrations of triglycerides, total cholesterol, low-density lipoprotein and insulin. The expression of adhesion molecules in platelets, aorta and monocyte-macrophage were enhanced by HFD. HFD also increased upstream proteins and their phosphorylated form in the aorta. In treatment groups, eugenosedin-A and atorvastatin improved HFD-induced hyperlipidaemia and levels of insulin. Eugenosedin-A reduced the upregulation of P-selectin, ICAM-1, ICAM-2, ICAM-3, VCAM, PECAM in platelets and inhibited E-selectin, ICAM-1, ICAM-2, ICAM-3, VCAM and PECAM protein levels in the aorta. Eugenosedin-A reduced the ACAT-1 protein expression of monocyte-macrophages. The expression of PKC alpha, MAPKs, IKK alpha and p65 and their phosphorylated form were reduced in treatment groups.Conclusions Taken together, hyperlipidaemia enhances the expression of adhesion molecules and ACAT-1 protein, and eugenosedin-A ameliorates those increases. Through inhibition of MAPK-and p-65-mediated NF-kappa B pathway, eugenosedin-A decreases the quantity of adhesion molecules.

In this study the effects of low-dose aspirin (5 mg/kg) on adhesion molecule and chemokine expression in a hyperlipidemic rat model. Six-week-old Sprague-Dawley (SD) rats were assigned to two control groups receiving either a regular diet or high-fat diet (HFD) and a treatment group fed HFD with 5 mg/kg aspirin for a 10-week period. Compared with the regular diet control group, the HFD control group had higher body weight, lower levels of high-density lipoprotein, higher concentrations of insulin, triglyceride, total cholesterol, and low-density lipoprotein, but no differences in blood glucose and glycated hemoglobin. The prothrombin time (PT) and activated partial thromboplastin time (aPTT) were clearly shortened in the HFD group. That group also had increased expression of intercellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3, vascular cell adhesion molecule (VCAM), platelet endothelial cell adhesion molecule (PECAM) and P-selectin in platelets and vascular adhesion protein-1 in lymphocyte and in aorta increased expressions of ICAM-1, ICAM-2, ICAM-3, VCAM, PECAM, E-selectin, monocyte chemoattractant protein-1 (MCP-1) and CCR2. The HFD rats also had increased PKC alpha, I kappa B kinase alpha (IKK alpha), p65, mitogen-activated protein kinases (MAPKs) (p38, c-Jun N-terminal kinases 1, extracellular signal-regulated kinase 1/2), and their phosphorylated forms. Low-dose aspirin improved HFD-induced hyperinsulinemia and hyperlipidemia, recovered PT and aPTT, inhibited upregulation of adhesion molecules and chemokines and reduced expression of PKC alpha, IKK alpha, p65, and MAPKs. Low-dose aspirin ameliorates HFD-induced hyperlipidemia and hyperinsulinemia, and prevents HFD-induced expression of adhesion molecules and chemokine formation.

Eugenosedin-A (Eu-A) effects on vascular endothelial dysfunction and oxidative stress in a hyperlipidemic rat model were investigated. Rats were randomly divided into four groups: two control groups and two treatment groups. The control rats received a regular diet or high fat diet (HFD); the treatment rats fed received an HFD with 5 mg/kg Eu-A or atorvastatin for 10 weeks. No changes in serotonin levels were observed in the four groups; norepinephrine levels were enhanced in the HFD group which was attenuated by Eu-A and atorvastatin. In the HFD group, the vascular reactivity was increased by vasoconstrictors (5-nonyloxytryptamine, 5-HT, and phenylephrine) and decreased by an endothelium-dependent vasorelaxant, carbachol. Protein levels of α1-adrenergic receptors (not 5-HT1B/2A), reactive oxygen species (ROS) p47(phox), p67(phox), and gp91(phox), and oxidative damage markers 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE) were increased, but endothelial nitric oxide synthase (eNOS), P-eNOS and vasodilator-stimulated phosphoprotein phosphorylation (P-VASP) were decreased. Catalase and superoxide dismutase (SOD-1 and SOD-2) proteins were increased, but glutathione peroxidase (GPx) was decreased in the aorta. Eu-A and atorvastatin reduced vasoconstrictor-induced aortic contractions that might be related to 5-HT1B/2A and α1-adrenergic receptors inhibitory activities. Eu-A and atorvastatin improved eNOS/P-eNOS, P-VASP, GPx, and malondialdehyde (MDA) levels, and decreased ROS and oxidative damage markers. Taken together, we suggest that Eu-A can ameliorate hyperlipidemia-induced vascular endothelial dysfunction and oxidative dysregulation.

BACKGROUND AND PURPOSESpinal muscular atrophy (SMA) is a progressive neuromuscular disease. Since disease severity is related to the amount of survival motor neuron (SMN) protein, up‐regulated functional SMN protein levels from the SMN2 gene are considered a major SMA drug‐discovery strategy. In this study, we investigated the possible effects of triptolide, a diterpene triepoxide purified from Tripterygium wilfordii Hook. F., as a new compound for increasing SMN protein.EXPERIMENTAL APPROACHThe effects and mechanisms of triptolide on the production of SMA protein were determined by cell‐based assays using the motor neuronal cell line NSC34 and skin fibroblasts from SMA patients. Wild‐type (Smn+/+SMN2−/−, C57BL/6) and SMA‐like (Smn−/−SMN2) mice were injected with triptolide (0.01 or 0.1 mg·kg−1·day−1, i.p.) and their survival rate and level of change in SMN protein in neurons and muscle tissue measured.KEY RESULTSIn NSC34 cells and human SMA fibroblasts, pM concentrations of triptolide significantly increased SMN protein expression and the levels of SMN complex component (Gemin2 and Gemin3). In human SMA fibroblasts, triptolide increased SMN‐containing nuclear gems and the ratio of full‐length transcripts (FL‐SMN2) to SMN2 transcripts lacking exon 7 (SMN2Δ7). Furthermore, in SMA‐like mice, triptolide significantly increased SMN protein levels in the brain, spinal cord and gastrocnemius muscle. Furthermore, triptolide treatment increased survival and reduced weight loss in SMA‐like mice.CONCLUSION AND IMPLICATIONSTriptolide enhanced SMN protein production by promoting SMN2 activation, exon 7 inclusion and increasing nuclear gems, and increased survival in SMA mice, which suggests triptolide might be a potential candidate for SMA therapy.

Objectives Eugenosedin-A has been found to ameliorate high-fat diet (HFD)-induced hyperglycaemia and hyperlipidaemia in C57BL/6J mice. This study aimed to investigate the mechanisms of action of eugenosedin-A on endothelial function and inflammation in hyperlipidaemic mice. Methods C57BL/6J mice were randomly divided into two control groups and two treatment groups. The control mice received either a regular diet or HFD, and the treatment groups were fed HFD with either 5 mg/kg eugenosedin-A or atorvastatin for eight weeks. Key findings Mice fed a HFD had higher concentrations of nitrate (NO) but not prostaglandin E2 (PGE2), increased tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) mRNA and inducible nitric oxide synthase (iNOS) proteins, but decreased endothelial nitric oxide synthase (eNOS) proteins. HFD-induced upregulation of iNOS is associated with p38, extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), PI3K and Akt/IKKα/p65. Eugenosedin-A and atorvastatin reduced HFD-induced TNF-α and IFN-γ mRNA, NO generation, upregulation of iNOS protein, and down-regulation of eNOS protein. Both agents inhibited p38, ERK, JNK and Akt/IKKα/p65 protein levels in the aorta. However, eugenosedin-A did not significantly reduce p38 in the liver. Conclusions Our results showed an association between obesity-induced inflammation and altered levels of TNF-α, IFN-γ, p38, ERK, JNK and Akt/IKKα/p65. Eugenosedin-A, like atorvastatin, could inhibit p38, ERK, JNK, Akt/IKKα/p65 proteins, as well as TNF-α and IFN-γ mRNA during the regulation of the obesity-induced inflammatory process.

Eugenosedin-B is able to block serotonin (5-HT) and alpha/beta receptors and to inhibit platelet aggregation. In Wistar rats, intravenous injections of eugenosedin-B (2.4, 7.2, 12 micromoL/kg) caused a dose-dependent decrease in blood pressure and heart rate. In contrast, intracisternal injection of eugenosedin-B (0.3, 0.03 micromoL) and an alpha2-antagonist yohimbine (0.03 micromoL) increased blood pressure and heart rate. Eugenosedin-B and yohimbine prevented hypotension induced by intracisternal injection of an alpha2-agonist clonidine (38 pmol). In in vitro experiments, eugenosedin-B (10, 10, 10 M) competitively antagonized norepinephrine-, clonidine-, and 5-HT (10 to 10 M)-induced vasocontractions in isolated rat aorta. It also competitively antagonized the isoproterenol (10 to 10 M)-induced positive inotropic effects in isolated rat atrium. These findings clearly suggest that eugenosedin-B possesses alpha1, alpha2, beta1, and 5-HT2A receptor blocking activities. In isolated rabbit ear artery sensitized with 16 mM K, eugenosedin-B antagonized 5-nonyloxytryptamine- and 5-HT-induced vasocontractions, indicating it also blocked 5-HT1B and 5-HT2A receptors. In radioligand-binding experiments, eugenosedin-B had significant binding affinities on alpha1, alpha2, beta1, 5-HT1B, and 5-HT2A receptors. In human platelets, eugenosedin-B inhibited epinephrine and 5-HT-induced aggregations. It also had competitive binding effects in human platelet with [H]yohimbine (alpha2), [H]ketanserin (5-HT2A). We conclude that hypotensive and vasorelaxant effects of eugenosedin-B can be attributed to its multiple actions on the blockade of 5-HT1B, 5-HT2A, alpha1/2 and beta1 receptors, and its ability to reduce platelet aggregation attributed to its blockade of alpha2 and 5-HT2A receptors.

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.

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)-α (0.01 mg/kg/300 μl) 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 μM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K+ channel, tetraethylammonium (10 mm); KATP channel, glibenclamide (1 μM); voltage-dependent K+ channel, 4-aminopyridine (100 μM); Ca2+-dependent K+ channel, charybdotoxin (0.1 μM) or apamin (1 μM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 μM); nitric-oxide (NO) synthase, Nω-nitro-l-arginine methyl ester (l-NAME, 100 μM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 μM). KMUP-3 (0.01-100 μM) 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 μM) induced expression of protein kinase A (PKA)ri and protein kinase G (PKG)1α 1β in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)ri.On the contrary, ODQ inhibited KMUP-3-induced expression of PKG1α 1β 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 Ca2+ influx in vitro, indicating the action mechanism to protect against TNF-α-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 μM). 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 μM), the sGC inhibitor ODQ (1 μM), the adenylyl cyclase (AC) inhibitor SQ 22536 (100 μM), and the prostaglandin inhibitor indomethacin (10 μM). 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 μM), the voltage-dependent K+ (KV) channel blocker 4-AP (100 μM), and Ca2+-dependent K+ (KCa) channel blockers apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM). In addition, elevated extracellular K+ (80 mM) interferes with KMUP-3- and KMUP-4-induced vasorelaxations. Preincubation with both agents (1 μM) 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 μM), 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.