Bin-Nan Wu

KMUP-1, a xanthine derivative, induces relaxation of guinea-pig isolated trachea: the role of the epithelium, cyclic nucleotides and K+ channels.

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 μM) 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 μM), 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 μM), nitric oxide synthase (Nω‐nitro‐L‐arginine methyl ester, 100 μM), K+ channels (tetraethylammonium, 10 mM), KATP channels (glibenclamide, 1 μM), voltage‐dependent K+ channels (4‐aminopyridine, 100 μM) and Ca2+‐dependent K+ channels (charybdotoxin, 0.1 μM or apamin, 1 μM). Both KMUP‐1 (10 μM) 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 μM, respectively. KMUP‐1 (0.01–10 μM) 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 μM) and an sGC inhibitor ODQ (10 μM), respectively. KMUP‐1 (10 μM) increased the expression of protein kinase A (PKARI) and protein kinase G (PKG1α1β) in a time‐dependent manner, but this was only significant for PKG after 9 h. Intratracheal administration of tumour necrosis factor‐α (TNF‐α, 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‐α, 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.

KMUP‐1 relaxes rabbit corpus cavernosum smooth muscle in vitro and in vivo: involvement of cyclic GMP and K+ channels

In isolated endothelium‐intact or denuded rabbit corpus cavernosum preconstricted with phenylephrine, KMUP‐1 (0.001 – 10 μM) caused a concentration‐dependent relaxation. This relaxation of KMUP‐1 was attenuated by endothelium removed, high K+ and pretreatments with a soluble guanylyl cyclase (sGC) inhibitor ODQ (1 μM), a NOS inhibitor L‐NAME (100 μM), a K+ channel blocker TEA (10 mM), a KATP channel blocker glibenclamide (1 μM), a voltage‐dependent K+ channel blocker 4‐AP (100 μM) and Ca2+‐dependent K+ channel blockers apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM). The relaxant responses of KMUP‐1 (0.01, 0.05, 0.1 μM) together with a PDE inhibitor IBMX (0.5 μM) had additive actions on rabbit corpus cavernosum smooth muscle (CCSM). KMUP‐1 (0.01 – 10 μM) 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 μM). 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. 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.

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

Background and purpose:  KMUP‐1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP‐1 on acute and chronic pulmonary artery hypertension (PAH) in rats.

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

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-α (TNF-α), a proinflammatory cytokine, increased the expression of inducible nitric-oxide synthase (iNOS) and NO production and decreased the expression of soluble guanylate cyclase α1 (sGCα1), soluble guanylate cyclase β1 (sGCβ1), 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-α-induced increases of iNOS expression and NO levels and reversed TNF-α-induced decreases of sGCα1, sGCβ1, and PKG expression. These results imply that cGMP enhancers could have anti-proinflammatory potential in TSMCs. TNF-α 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-PGF1α (stable PGI2 metabolite). Dexamethasone and N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398; a selective COX-2 inhibitor) attenuated TNF-α-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-α. It is suggested that TNF-α-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-α-induced expression of iNOS in TSMCs, involving the sGC/cGMP/PKG expression pathway but without the involvement of COX-2.

Antioxidant eugenosedin-A protects against lipopolysaccharide-induced hypotension, hyperglycaemia and cytokine immunoreactivity in rats and mice

Eugenosedin‐A has been demonstrated to possess α/β‐adrenoceptor and serotonergic receptor blocking activities. We have investigated by what mechanisms eugenosedin‐A prevents lipopolysaccharide (LPS)‐induced hypotension, vascular hyporeactivity, hyperglycaemia, oxidative injury or inflammatory cytokines formation in rats. Intravenous administration of eugenosedin‐A, trazodone, yohimbine (1 mg kg−1), aminoguanidine or ascorbic acid (15 mg kg−1) normalized LPS (10 mg kg−1)‐induced hypotension. Pretreatment with eugenosedin‐A or the other agents 30 min before LPS injection reduced aortic hyporeactivity. LPS‐induced increases in plasma interleukin‐1β (IL‐β), IL‐6, interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and blood glucose levels were significantly inhibited by eugenosedin‐A (1 mg kg−1, i.v.). The same dose of trazodone, a chloropiperazinylbenzene‐type antidepressant, and yohimbine, an α2‐adrenoceptor antagonist, reduced IL‐1β and TNF‐α, but it could not inhibit hyperglycaemia. Aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, and ascorbic acid, an antioxidant, decreased IL‐1β, TNF‐α contents and hyperglycaemia. Eugenosedin‐A and the other agents inhibited Fe2+‐ascorbic acid‐induced peroxidation in rat cortex, indicating that those agents had antioxidant effects, with the exception of aminoguanidine. In free radical scavenged experiments, eugenosedin‐A and ascorbic acid eliminated peroxyl radicals. All test agents inhibited the LPS‐induced increase of malondialdehyde (MDA) content in rat brain homogenates. When mice were administered an intraperitoneal injection of LPS alone, mortality occurred from 4 to 16 h, after which time all were dead. However, eugenosedin‐A significantly prolonged the survival time after LPS injection, suggesting that eugenosedin‐A protected against LPS‐induced cardiovascular dysfunction, hyperglycaemia, tissue injury and inflammatory cytokine production. This was attributable mainly to the antioxidant and peroxyl radical scavenged effects of eugenosedin‐A, and which may be, at least in part, due to its blockade on α/β‐adrenergic and serotonergic receptors.

Baicalin, a flavonoid from Scutellaria baicalensis Georgi, activates large-conductance Ca2+-activated K+ channels via cyclic nucleotide-dependent protein kinases in mesenteric artery.

Baicalin isolated from Scutellaria baicalensis is a traditional Chinese herbal medicine used for cardiovascular dysfunction. The ionic mechanism of the vasorelaxant effects of baicalin remains unclear. We investigated whether baicalin relaxes mesenteric arteries (MAs) via large-conductance Ca2+-activated K+ (BK(Ca)) channel activation and voltage-dependent Ca2+ channel (VDCC) inhibition. The contractility of MA was determined by dual wire myograph. BK(Ca) channels and VDCCs were measured using whole-cell recordings in single myocytes, enzymatically dispersed from rat MAs. Baicalin (10-100 microM) attenuated 80 mM KCl-contracted MA in a concentration-related manner. L-NAME (30 microM) and indomethacin (10 microM) little affected baicalin (100 microM)-induced vasorelaxations. Contractions induced by iberiotoxin (IbTX, 0.1 microM), Bay K8644 (0.1 microM) or PMA (10 microM) were abolished by baicalin 100 microM. In MA myocytes, baicalin (0.3-30 microM) enhanced BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) currents were abolished by IbTX (0.1 microM). Baicalin-mediated (30 microM) BK(Ca) current activation was significantly attenuated by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Perfusate with PMA (0.1 microM) abolished baicalin-enhanced BK(Ca) currents. Additionally, baicalin (0.3-30 microM) reduced the amplitude of VDCC currents in a concentration-dependent manner and abolished VDCC activator Bay K8644-enhanced (0.1 microM) currents. Baicalin produced MA relaxation by activating BK(Ca) and inhibiting VDCC channels by endothelium-independent mechanisms and by stimulating the cGMP/PKG and cAMP/PKA pathways.

KMUP‐1 attenuates isoprenaline‐induced cardiac hypertrophy in rats through NO/cGMP/PKG and ERK1/2/calcineurin A pathways

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.

A Highly Selective β1-adrenergic Blocker with Partial β2-agonist Activity Derived from Ferulic Acid, an Active Component of ligusticum wallichii Franch

Short-term injection of ferulinolol (0.1, 0.5, and 1.0 mg/kg, i.v.) produced dose-dependent bradycardia responses in pentobarbital-anesthetized Wistar rats, whereas it had no significant effects on the blood pressure. Ferulinolol markedly inhibited the tachycardia effects induced by (-)isoproterenol but did not show any blocking effect on the arterial pressor responses induced by (-)phenylephrine. These findings clearly suggested that ferulinolol had a beta-adrenergic blocking activity; nevertheless, it did not involve an alpha-adrenergic blocking action. In isolated guinea pig tissues, ferulinolol competitively antagonized (-)isoproterenol-induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses. The parallel shift to the right of the concentration-response curve of (-)isoproterenol suggested that ferulinolol was a beta-adrenoceptor-competitive antagonist. The apparent pA2 values for ferulinolol on right atria, left atria, and trachea were 7.62 +/- 0.05, 7.54 +/- 0.01, and 6.28 +/- 0.11, respectively. Ferulinolol was more potent on the atria than on tracheal tissues, demonstrating that it possessed beta1-adrenoceptor selectivity. The intrinsic sympathomimetic activity (ISA) of ferulinolol and propranolol were determined on isolated atria and trachea from reserpine-treated guinea pig. Propranolol caused significantly negative inotropic and chronotropic effects at > or =1 microM, whereas ferulinolol possessed fewer cardiodepressant activities than propranolol. In reserpine-treated tracheal strips, ferulinolol produced dose-dependent relaxant responses, but propranolol was without effectiveness. Preincubating the preparations with ICI 118,551 (0.1, 1.0, and 10 nM), a beta2-adrenoceptor antagonist, significantly shifted the concentration-relaxation curves of ferulinolol to a region of higher concentrations. These results implied that ferulinolol had a partial beta2-agonist activity. Further, binding characteristics of ferulinolol and various beta-adrenoceptor antagonists were evaluated in [3H]CGP-12177 binding to rat ventricular or lung membranes. The Ki values of ferulinolol, atenolol, metoprolol, and (-)propranolol were 103, 262, 123, and 0.23 nM, respectively, in ventricular membranes, and 2,412, 7,539, 2,186, and 0.72 nM, respectively, in lung membranes. In conclusion, ferulinolol was found to be a highly selective beta1-adrenoceptor antagonist with partial beta2-agonist activity but was devoid of alpha-adrenoceptor blocking action.

Eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kappaB AND AP-1 through inhibition of MAPKS and AKT/IkappaBalpha signaling pathways in macrophages.

Eugenol and isoeugenol, two components of clover oil, have been reported to possess several biomedical properties, such as anti-inflammatory, antimicrobial and antioxidant effects. This study aims to examine the anti-inflammatory effects of eugenol, isoeugenol and four of their derivatives on expression of inducible nitric oxide synthase (iNOS) activated by lipopolysaccharide (LPS) in mouse macrophages (RAW 264.7), and to investigate molecular mechanisms underlying these effects. We found that two derivatives, eugenolol and glyceryl-isoeugenol, had potent inhibitory effects on LPS-induced upregulation of nitrite levels, iNOS protein and iNOS mRNA. In addition, they both suppressed the release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induced by LPS. Moreover, they both attenuated the DNA binding of NF-κB and AP-1, phosphorylation of inhibitory κBα (IκBα), and nuclear translocation of p65 protein induced by LPS. Finally, we demonstrated that glyceryl-isoeugenol suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK, whereas eugenolol suppressed the phosphorylation of ERK1/2 and p38 MAPK. Taken together, these results suggest that that eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-κB and AP-1 through inhibition of MAPKs and Akt/IκBα signaling pathways. Thus, this study implies that eugenolol and glyceryl-isoeugenol may provide therapeutic benefits for inflammatory diseases.

KMUP-1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide-dependent protein kinases

This study investigated whether KMUP‐1, a synthetic xanthine‐based derivative, augments the delayed‐rectifier potassium (KDR)‐ 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 μM) had no effect on the KDR 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 μM), an adenylate cyclase inhibitor (SQ 22536 10 μM), competitive antagonists of cGMP and cAMP (Rp‐cGMP, 100 μM and Rp‐cAMP, 100 μM), 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 μM), and nearly abolished by a calcium channel blocker (nifedipine, 1 μM). 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.