Baicalein, isolated from Scutellaria baicalensis, protects against endothelin-1-induced pulmonary artery smooth muscle cell proliferation via inhibition of TRPC1 channel expression.

ETHNOPHARMACOLOGICAL RELEVANCE: We investigated the antiproliferative effects of baicalein, isolated from Scutellaria baicalensis (Huang-qin), on ET-1-mediated pulmonary artery smooth muscle cells (PASMCs) proliferation and the mechanisms underlying these effects. MATERIALS AND METHODS: Intrapulmonary artery smooth muscle cells were isolated and cultured from female Sprague-Dawley rats and used during passages 3-6. The proliferation of PASMCs was quantified by cell counting and XTT assay. The protein expression of TRPC1 and PKCα were determined by western blotting. The cell cycle pattern was assayed by flow cytometry. The intracellular calcium concentrations ([Ca(2+)](i)) were measured using the fluorescent indicator fura-2-AM and flow cytometry. RESULTS: Baicalein (0.3-3 µM) inhibited PASMCs proliferation, promoted cell cycle progression, enhanced [Ca(2+)](i) levels, increased capacitative Ca(2+) entry (CCE), upregulated the canonical transient receptor potential 1 (TRPC1) channel and membrane protein kinase Cα (PKCα) expression induced by ET-1 (0.1 µM). The PKC activator PMA (1 µM) reversed the inhibitory effects of baicalein on ET-1-induced upregulation of TRPC1 expression and S phase accumulation, while the PKC inhibitor chelerythrine (1 µM) potentiated baicalein-mediated G(2)/M phase arrest and TRPC1 channel inhibition. CONCLUSION: Our findings suggest that baicalein protects against ET-1-induced PASMCs proliferation via modulation of the PKC-mediated TRPC channel.

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

Eugenosedin-A has been demonstrated to possess alpha/beta-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-1beta (IL-beta), IL-6, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) 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 alpha(2)-adrenoceptor antagonist, reduced IL-1beta and TNF-alpha, but it could not inhibit hyperglycaemia. Aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, and ascorbic acid, an antioxidant, decreased IL-1beta, TNF-alpha contents and hyperglycaemia. Eugenosedin-A and the other agents inhibited Fe(2+)-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 alpha/beta-adrenergic and serotonergic receptors.

San-Huang-Xie-Xin-Tang reduces lipopolysaccharides-induced hypotension and inflammatory mediators.

San-Huang-Xie-Xin-Tang (SHXT) is a traditional Chinese medicinal formula containing Coptidis rhizoma, Scutellariae radix and Rhei rhizoma. The present study aimed to determine the preventive effects of standardized SHXT on lipopolysaccharides (LPS)-induced arterial hypotension, protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), cytokines formation and prostaglandin E2 (PGE2) production. LPS-induced activation of iNOS has been recognized to increase cytokines and nitric oxide, some of them play predominant roles in sepsis. Intravenous injection of LPS (10 mg/kg) caused a marked decrease of the mean arterial pressure in normotensive rats. However, the LPS-induced arterial hypotension was inhibited by SHXT (0.01 and 0.03 g/kg), when it was given 30 min before LPS. Moreover, plasma level of cytokines and PGE2 were lowered by SHXT. In RAW 264.7 cells, SHXT (20-200 microg/ml) dose-dependently inhibited LPS (1 microg/ml)-induced iNOS and COX-2 expression, and it also significantly decreased LPS-induced cytokines in a dose-dependent manner. In conclusion, our data suggest that SHXT prevented LPS-induced arterial hypotension, which might be mediated through its inhibition activities on the expression of iNOS and COX-2, cytokines formation and PGE2 production. Therefore, its protection activity against LPS-induced arterial hypotension and inflammatory mediators release might be beneficial in the treatment of endotoxin shock and/or associated inflammation.

Urgosedin inhibits hypotension, hypoglycemia, and pro-inflammatory mediators induced by lipopolysaccharide.

Urgosedin is a newly synthesized compound especially with serotonergic and alpha-adrenergic blocking actions. In rat isolated thoracic aorta, urgosedin competitively antagonized norepinephrine-, clonidine-, and serotonin-induced vasocontractions in a concentration-dependent manner. In radioligand binding experiments, urgosedin had significant binding affinities on alpha1/alpha2, 5-HT1A, 5-HT1B and 5-HT2A receptors. Intravenous injection of lipopolysaccharide (LPS) produced a biphasic hypotension in normotensive rats. Although intravenous injection of urgosedin caused minor depressor actions in the normotensive Wistar rat, urgosedin significantly attenuated the secondary prolonged hypotension produced by LPS. The plasma levels of cytokines (IL-1beta, IL-6, TNF-alpha, and IFN-gamma) and hypoglycemia induced by LPS were also reduced by urgosedin. Moreover, the acute survival rates (350 minutes) of endotoxic shock increased from 0% (LPS group) to 100% in the groups pretreated with urgosedin. In RAW264.7 cells, urgosedin inhibited LPS-induced inducible nitric oxide synthase (iNOS) expression. In conclusion, our data suggest that urgosedin was a newly potent serotonergic and mild alpha-adrenergic blocking agent. Its prevention of LPS-induced hypotension and hypoglycemia might partially mediate through its inhibition activities on the iNOS expression and cytokines formation. Urgosedin might be an effective pharmacological agent against LPS-induced hypotension, hypoglycemia, and the formation of pro-inflammatory mediators.

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 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.

Hypotensive effects of eugenosedin-A with serotonin, alpha- and beta-adrenoceptor antagonistic activities in spontaneously hypertensive and normotensive rats.

Eugenosedin-A is a newly synthesized compound with special serotonergic, alpha- and beta1-adrenergic blocking actions. Intravenous injection of eugenosedin-A significantly caused dose-dependent decreases in the mean arterial blood pressure and heart rate in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). The effects of eugenosedin-A-decreased blood pressure and heart rate in SHR were more potent than in WKY. In in vitro experiments, eugenosedin-A competitively antagonized the serotonin-, norepinephrine- and clonidine-induced vasocontraction in a concentration-dependent manner in isolated thoracic aorta of WKY and SHR. We also observed that eugenosedin-A competitively antagonized the isoproterenol-induced positive inotropic effects in a concentration-dependent manner in the isolated left atrium of WKY and SHR. These findings clearly suggested that eugenosedin-A possesses alpha1/alpha2, beta1 and 5-HT2A receptor-blocking activities. The order of pA2 values in isolated tissues of WKY was 5-HT2A > alpha1/alpha2 > beta1. However, the order of pA2 values in isolated tissues of SHR was alpha1/alpha2 > 5-HT2A > beta1. Similarly, we found that the in vitro functional activity of eugenosedin-A is quite different between WKY and SHR. On the other hand, in the isolated rabbit ear artery sensitized with 16 mmol/l K+, eugenosedin-A antagonized 5-nonyloxytryptamine- and serotonin-induced vasocontractions, indicating that it also blocked 5-HT1B and 5-HT2A receptors. In radioligand binding experiments, eugenosedin-A had significant binding affinities on alpha1/alpha2, beta1, 5-HT1B and 5-HT2A receptors. Finally, we suggest that the hypotensive effects of eugenosedin-A can be attributed to its multiple actions on the blockade of 5-HT1B, 5-HT2A, alpha and beta1 receptors in both WKY and SHR strains.

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

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.