Effects of 3′,4′‐dihydroxyflavonol on vascular contractions of rat aortic rings

1. 3′,4′‐Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti‐oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions. 2. Contractile and relaxation responses were determined in rat endothelium‐denuded aortic rings mounted in organ baths. In addition, the phosphorylation of myosin light chain (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)‐potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI‐17) was determined using western blot analaysis. Levels of GTP RhoA, as a marker of RhoA activation, were also measured. 3. Cumulative addition of increasing concentrations of NaF (3.0–12.0 mmol/L) or U46619 (1.0–1000 nmol/L) concentration‐dependently increased vascular tension. These responses were inhibited by pretreatment of aortic rings with DiOHF (10, 30 or 100 μmol/L), which dose‐dependently decreased vascular contractions induced by 8.0 mmol/L NaF, 30 nmol/L U46619, 0.1 μmol/L phenylephrine and 50 mmol/L KCl. 4. The K⁺ channel blockers 4‐aminopyridine (3 mmol/L), charybdotoxin (10 nmol/L), apamin (500 nmol/L) and glibenclamide (10 μmol/L) had no effect on vascular relaxation induced by DiOHF (1–30 μmol/L). 5. At 30 μmol/L, DiOHF decreased the activation of RhoA and subsequent phosphorylation of MYPT1, CPI‐17 and MLC₂₀ to almost basal levels. 6. In conclusion, DiOHF decreases vascular contraction at least partly by inhibition of the RhoA/Rho‐kinase pathway in rat endothelium‐denuded aorta. These results suggest that DiOHF may have therapeutic potential in the treatment of cardiovascular diseases.     

Honokiol attenuates vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway in rat aortic rings

Objectives Honokiol is a small‐molecule polyphenol isolated from the species Magnolia obovata. We hypothesized that honokiol attenuated vascular contractions through the inhibition of the RhoA/Rho‐kinase signalling pathway. Methods Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. Phosphorylation of 20 kDa myosin light chains (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)‐potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase (MLCP) of 17 kDa (CPI17) were examined by immunoblot. We also measured the amount of guanosine triphosphate RhoA as a marker for RhoA activation. Key findings Pretreatment with honokiol dose‐dependently inhibited the concentration–response curves in response to sodium fluoride (NaF) or thromboxane A₂ agonist U46619. Honokiol decreased the phosphorylation levels of MLC₂₀, MYPT1_Thr855 and CPI17_Thr38 as well as the activation of RhoA induced by 8.0 mm NaF or 30 nm U46619. Conclusions These results demonstrated that honokiol attenuated vascular contraction through the inhibition of the RhoA/Rho‐kinase signalling pathway.     

17β-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway

We hypothesized that 17β-estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway. Rat aortic rings were contracted with cumulative addition of U46619, NaF, KCl or PDBu 30 min after pretreatment with 17β-estradiol (10, 30, and 100 μM) or vehicle. We measured the amount of GTP RhoA and the level of phosphorylation of the myosin light chain (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Pretreatment with 17β-estradiol dose-dependently inhibited the concentration-response curves in response to U46619, NaF or KCl, but not to PDBu. 17β-Estradiol decreased not only the level of phosphorylation of MYPT1^Thr855 and CPI17^Thr38 as well as MLC₂₀, but also the activity of RhoA induced by U46619 or NaF. However, 17β-estradiol did not affect the level of phosphorylation of CPI17 induced by PDBu. 17β-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway.     

Effects of glyceollin I on vascular contraction in rat aorta

The present study was undertaken to investigate the molecular mechanisms by which glyceollin I inhibits vascular contraction in rat aorta. Rat aortic rings were treated with either glyceollin I or vehicle when vascular contraction reached plateaus. We measured the activity of GTP-RhoA and Rho GTPase-activating protein (RhoGAP) and the phosphorylation level of the myosin light chain (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1), and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Glyceollin I reduced vascular contraction whether endothelium is present or denuded. Glyceollin I reduced vascular contraction induced by NaF, U46619, phenylephrine, or PDBu. Blockers of K⁺ channels did not affect the vasorelaxation induced by glyceollin I. Glyceollin I reduced activation of RhoA as well as phosphorylation level of MLC₂₀. Glyceollin I also reduced phosphorylation of MYPT1 and CPI17 induced by NaF but not PDBu. However, glyceollin I had no direct effect on RhoGAP activation in vitro. Glyceollin I reduced vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.     

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 µM) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC₂₀), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC₂₀ phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619-or NaF-induced phosphorylation of MYPT1^Thr855 and CPI17^Thr38, the downstream effectors of Rho-kinase. In regards to the Ca²⁺-free solution, flavone inhibited the phosphorylation of MYPT1^Thr855 and CPI17^Thr38, as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.     

Effects of gomisin A on vascular contraction in rat aortic rings

Gomisin A (GA) is an active ingredient of the fruits of Schisandra chinensis which has been widely used as a tonic in traditional Korean medicine. GA induces not only endothelium-dependent but also endothelium-independent relaxation in an isolated rat's thoracic aorta. This study was aimed to investigate the molecular mechanism by which GA induces endothelium-independent vasorelaxation. Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. We measured the amount of GTP RhoA as well as the phosphorylation level of 20 kDa myosin light chains (MLC??), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17). Pretreatment with GA dose-dependently inhibited the concentration-response curves in response to sodium fluoride (NaF) or thromboxane A(2) agonist U46619, but not to phorbol 12, 13-dibutyrate (PDBu). GA decreased the activation of RhoA as well as the phosphorylation level of MLC??, MYPT1(Thr???), and CPI17 induced by 8.0 mM NaF or 30 nM U46619. However, K+ channel blockers such as glibenclamide, apamin, or charybdotoxin did not affect the vascular relaxation induced by GA. Furthermore, GA did not affect the level of phosphorylation of CPI17 induced by PDBu. GA reduces vascular contraction through inhibition of RhoA/Rho-kinase pathway in endothelium-denuded rat aorta.     

MAGNOLOL DEPRESSES UROTENSIN-II-INDUCED CELL PROLIFERATION IN RAT CARDIAC FIBROBLASTS

• 1 Accumulating evidence suggests that oxidative stress plays a key role in the development of cardiac fibrosis. Urotensin‐II (U‐II) has been reported to play an important role in cardiac remodelling and fibrosis. Recently, we demonstrated the involvement of reactive oxygen species (ROS) production in U‐II‐induced cardiac fibroblast proliferation. Magnolol is an anti‐oxidant compound extracted from the cortices of Magnolia officinalis. Thus, it is feasible that magnolol may attenuate cardiac fibroblast proliferation by inhibiting ROS production. Therefore, the aims of the present study were to determine whether magnolol alters U‐II‐induced cell proliferation and to identify the putative underlying signalling pathways in rat cardiac fibroblasts.
• 2 Cultured rat cardiac fibroblasts were pretreated with magnolol (1, 3 and 10 µmol/L) for 30 min, followed by exposure to U‐II (30 nmol/L) for 24 h, after which cell proliferation and endothelin‐1 (ET‐1) protein secretion was examined. The effects of magnolol on U‐II‐induced ROS formation and extracellular signal‐regulated kinase (ERK) phosphorylation were examined to elucidate the intracellular mechanisms by which magnolol affects cell proliferation and ET‐1 expression.
• 3 Urotensin‐II (30 nmol/L) stimulated cell proliferation, ET‐1 protein secretion and ERK phosphorylation, all of which were inhibited by magnolol (10 µmol/L). Pretreatment of cardiac fibroblasts with N‐acetylcysteine (5 mmol/L) for 30 min prior to exposure to U‐II resulted in inhibition of U‐II increased ROS formation. Similar effects were observed with 10 µmol/L magnolol.
• 4 In conclusion, the results suggest that magnolol inhibits cardiac fibroblast proliferation by interfering with ROS generation. Thus, the present study provides important new insights into the molecular pathways involved, which may contribute to our understanding of the effects of magnolol on the cardiovascular system.

     

Inhibition of cGMP phosphodiesterase 5 suppresses matrix metalloproteinase‐2 production in pulmonary artery smooth muscles cells

1. It has been shown that the beneficial effects of phosphodiesterase (PDE) 5 inhibition on pulmonary hypertension (PH) are associated with the induction of vascular relaxation and suppression of the proliferation of pulmonary artery smooth muscle cells (PASMC). In the present study, we investigated whether PDE5 inhibition affects the production and/or secretion of matrix metalloproteinases (MMPs) in PASMC, resulting in extracellular matrix remodelling in the pulmonary vasculature and, thus, the development of PH. 2. Primary cultured PASMC were stimulated with endothelin (ET)‐1 and MMP‐2 production and RhoA activation were then determinded using gelatin zymography and a GTP‐bound RhoA assay, respectively. The effects of the selective PDE5 inhibitor sildenafil and subsequent protein kinase G‐specific inhibitor Rp‐8Br‐cGMPs on MMP‐2 production and RhoA activation were further exmamined. 3. Endothelin‐1 (1–1000 nmol/L) concentration‐dependently stimulated MMP‐2 production and/or secretion in primary cultured PASMC, with 100 nmol/L ET‐1 causing a 2.41‐fold increase in MMP‐2 production compared with control (P < 0.01). This increase in MMP‐2 production was accompanied by RhoA activation, which was abolished by preincubation of cells with 10 μmol/L Y27632, an inhibitor of Rho‐associated kinase (ROCK). Furthermore, 10 μmol/L Y27632 abolished the ET‐1‐induced production of MMP‐2. 4. The selective PDE5 inhibitor sildenafil (0.1–1 μmol/L) concentration‐dependently reduced the increased MMP‐2 production induced by 100 nmol/L ET‐1. Specifically, in the presence of 1 μmol/L sildenafil, the 100 nmol/L ET‐1‐induced increase in MMP‐2 production was only increased 1.3‐fold over that of the control (P < 0.01 vs 100 nmol/L ET‐1‐stimulated cells). 5. Suppression of RhoA activation was found to mediate the inhibitory effect of sildenafil on ET‐1‐induced increases in MMP‐2 production. Furthermore, the protein kinase G‐specific inhibitor Rp‐8Br‐cGMPs reversed the inhibitory effects of sildenafil on RhoA activation and MMP‐2 production. 6. The results of the present study indicate that PDE5 inhibition suppresses RhoA/ROCK‐mediated MMP‐2 production by PASMC, which may contribute to the regulation of pulmonary vascular remodelling. Thus, PDE5 inhibition may benefit patients with PH through multiple mechanisms of action.     

ENDOTHELIAL DYSFUNCTION EXACERBATES THE IMPAIRMENT OF RELAXATION BY LYSOPHOSPHATIDYLCHOLINE IN PORCINE CORONARY ARTERY

1. Current evidence suggests that lysophosphatidylcholine (LPC), a component found in oxidized low-density lipoprotein (Ox-LDL), inhibits endothelium-dependent relaxation (EDR) mediated by endothelium-derived relaxing factor (EDRF) and endothelium-derived hyperpolarizing factor (EDHF). An objective of the present study was to characterize the roles of the different elements of EDR in LPC-induced impairment within the porcine coronary artery. Concomitantly, we sought to determine whether impairment of one component of EDR would increase the sensitivity of the endothelium to LPC. 2. Bradykini. (0.1 nmol/L-0.3 μmol/L) relaxed U46619 (30 nmol/L)-precontracted porcine coronary artery rings in a concentration-dependent manner. A reduction in the bradykinin-elicited response was observed in N^G-nitro-L-arginine methyl ester (L-NAME; 300 μmol/L)- and ouabain (50 μmol/L)-treated rings. Pretreatment with LPC (20 μmol/L), which on its own had no effect on normal endothelial relaxation, resulted in further inhibition of EDRF- and EDHF-induced relaxations. 3. Our results demonstrate that EDRF and EDHF are the primary mediators of EDR in the porcine coronary artery. Our data also show that while a low concentration of LPC (20 μmol/L) does not impair EDR, it can evoke vascular dysfunction following blockade of either the effects of EDRF or EDHF. Therefore, these data suggest that the partially damaged vascular endothelium could be more sensitive to threshold levels of this atherogenic phospholipid.     

DIRECT EFFECT OF DEXMEDETOMIDINE ON RAT ISOLATED AORTA INVOLVES ENDOTHELIAL NITRIC OXIDE SYNTHESIS AND ACTIVATION OF THE LIPOXYGENASE PATHWAY

• 1 The aims of the present in vitro study were to examine the roles of pathways associated with arachidonic acid metabolism in dexmedetomidine‐induced contraction and to determine which endothelium‐derived vasodilators are involved in the endothelium‐dependent attenuation of vasoconstriction elicited by dexmedetomidine.
• 2 Dexmedetomidine (10^?9–10^?6 mol/L) concentration–response curves were constructed in: (i) aortic rings with no drug pretreatment; (ii) endothelium‐denuded aortic rings pretreated with either 2 × 10^?5 mol/L quinacrine dihydrochloride, 10^?5 mol/L nordihydroguaiaretic acid (NDGA), 3 × 10^?5 mol/L indomethacin or 10^?5 mol/L fluconazole; and (iii) endothelium‐intact aortic rings pretreated with either 5 × 10^?5 mol/L N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME), 10^?5 mol/L fluconazole, 10^?5 mol/L indomethacin, 10^?5 mol/L glibenclamide, 5 × 10^?3 mol/L tetraethylammonium or 5 × 10^?5 mol/L l ‐NAME plus rauwolscine (10^?5, 10^?6 mol/L). The production of nitric oxide (NO) metabolites was determined in human umbilical vein endothelial cells treated with dexmedetomidine.
• 3 Quinacrine dihydrochloride, NDGA and indomethacin attenuated the dexmedetomidine‐induced contraction of endothelium‐denuded rings. Dexmedetomidine (10^?7–10^?6 mol/L)‐induced contractions of endothelium‐denuded rings were enhanced compared with those of endothelium‐intact rings, as were dexmedetomidine‐induced contractions of endothelium‐intact rings pretreated with l ‐NAME or tetraethylammonium. Rauwolscine attenuated dexmedetomidine‐induced contractions in endothelium‐intact rings pretreated with l ‐NAME. Dexmedetomidine (10^?6 mol/L) was found to activate NO production.
• 4 Taken together, the results indicate that dexmedetomidine‐induced contraction of aortic rings involves activation of the lipoxygenase and cyclo‐oxygenase pathways and is attenuated by increased NO production following stimulation of endothelial α₂‐adrenoceptors by dexmedetomidine.

     

Vasorelaxation by Samhwangsasim-tang, an herb medicine, is associated with decreased phosphorylation of the myosin phosphatase target subunit

Samhwangsasim-tang (SST) is a widely used herbal medicine with vasodilatory actions in oriental countries. We hypothesized that SST modulates vascular contractility by decreasing phosphorylation of the myosin phosphatase target subunit.

Rat aortic ring preparations were mounted in organ baths and subjected to contractions or relaxations. Phosphorylation of 20 kDa myosin light chains (MLC₂₀) and MYPT1, a target subunit of myosin phosphate 1, were examined with immunoblots. SST relaxed aortic ring preparations precontracted with phenylephrine whether endothelium was intact or denuded. Treatment of aortic rings with N^ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of endothelial nitric oxide synthase or methylene blue, an inhibitor of guanylyl cyclase, did not affect the relaxing action of SST. Furthermore, SST inhibited vascular contractions induced by NaF or phenylephrine, but not by phorbol dibutyrate. SST also decreased vascular tension precontracted by 8.0 mmol/L NaF or 1.0 μmol/L phenylephrine, but not by 1.0 μmol/L phorbol dibutyrate. In vascular strips, SST decreased the phosphorylation level of both MLC₂₀ and MYPT1 induced by 8.0 mmol/L NaF.

In conclusion, SST inhibited vascular contraction by decreasing phosphorylation of the myosin phosphatase target subunit.     

Trimebutine maleate relaxes the isolated rat thoracic aorta: The role of nitric oxide and L‐type calcium channels

Trimebutine maleate (TMB), a widely prescribed drug for functional gastrointestinal disorders, has been reported to regulate smooth muscle contractility by modulating multiple ion channel activities in the gastrointestinal tract. However, its action on isolated aorta has not yet been reported. The aim of the present study was to evaluate in vitro vasorelaxant properties and the underlying pharmacological mechanisms of TMB in isolated rat thoracic aortic rings. Vascular activity experiments were performed on thoracic aorta isolated from Sprague‐Dawley rats in vitro, including endothelium‐intact and endothelium‐denuded aortic rings. TMB (10^?10‐10^?5 mol/L) induced relaxation in endothelium‐intact aortic rings precontracted by phenylephrine with a potency similar to that of carbachol. TMB‐induced relaxation was not altered by glibenclamide and atropine in endothelium‐intact aortic rings. However, L‐NAME and endothelium denudation significantly reduced but not completely reversed the vasorelaxant effect of TMB. Also, TMB‐induced relaxation wasn't affected by diclofenac in endothelium‐intact aortic rings. TMB at 10^?5 mol/L significantly reduced the CaCl₂‐induced contractions in endothelium‐intact aortic rings stimulated with KCl, but not stimulated with phenylephrine under Ca²⁺free conditions. Moreover, TMB at 10^?5 mol/L effectively attenuated Bay‐K8644‐induced contractions in aortic rings. These results suggest that TMB‐induced relaxation was mediated by both endothelium‐dependent and endothelium‐independent manner in isolated rat thoracic aorta. The mechanism of TMB‐induced relaxation at low concentrations is partially related to NO‐ and endothelium‐dependent but unrelated to prostanoids formation. However, inhibition of Ca²⁺ influx through voltage‐operated calcium channels and L‐type Ca²⁺channel blocking effect appears to be involved in the mechanism of vasorelaxant effect of TMB at high concentrations.     

The Relaxant Effect of Propofol on Isolated Rat Intrapulmonary Arteries

Propofol is a widely used anesthetic. Many studies have shown that propofol has direct effects on blood vessels, but the precise mechanism is not fully understood. Secondary intrapulmonary artery rings from male rats were prepared and mounted in a Multi Myograph System. The following constrictors were used to induce contractions in isolated artery rings: high K⁺ solution (60 mmol/L); U46619 solution (100 nmol/L); 5-hydroxytryptamine (5-HT; 3 µmol/L); or phenylephrine (Phe; 1 µmol/L). The relaxation effects of propofol were tested on high K⁺ or U46619 precontracted rings. Propofol also was added to induce relaxation of rings preconstricted by U46619 after pretreatment with the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME). The effects of propofol on Ca²⁺ influx via the L-type Ca²⁺ channels were evaluated by examining contraction-dependent responses to CaCl₂ in the absence or presence of propofol (10 to 300 µmol/L). High K⁺ solution and U46619 induced remarkable contractions of the rings, whereas contractions induced by 5-HT and Phe were weak. Propofol induced dose-dependent relaxation of artery rings precontracted by the high K⁺ solution. Propofol also induced relaxation of rings precontracted by U46619 in an endothelium-independent way. Propofol at different concentrations significantly inhibited the Ca²⁺-induced contractions of pulmonary rings exposed to high K⁺-containing and Ca²⁺-free solution in a dose-dependent manner. Propofol relaxed vessels precontracted by the high K⁺ solution and U46619 in an endothelium-independent way. The mechanism for this effect may involve inhibition of calcium influx through voltage-operated calcium channels (VOCCs) and receptor-operated calcium channels (ROCCs).     

Flavone inhibits vascular contraction by decreasing phosphorylation of the myosin phosphatase target subunit

1. Flavonoids modulate vascular tone through an endothelium-dependent or -independent mechanism. Although a few mechanisms for endothelium-independent relaxation have been suggested, such as interference with protein kinase C or cAMP or cGMP phosphodiesterase, the inhibition of Ca(2+) release from intracellular stores or Ca(2+) influx from extracellular fluids, the mode of action of flavonoids remains elusive. 2. We hypothesized that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the myosin phosphatase target subunit (MYPT1). 3. Rat aortic rings were denuded of endothelium, mounted in organ baths and contracted with U46619, a thromboxane A(2) analogue. 4. Flavone dose-dependently inhibited the U46619-induced contractile response and myosin light chain (MLC(20)) phosphorylation. At 10(-7) mol/L, U46619 induced vascular contraction with the concomitant phosphorylation of MYPT1 at Thr855, but not at Thr697. Incubation with flavone (100 or 300 micromol/L) for 30 min attenuated the phosphorylation of MYPT1(Thr855), but not MYPT1(Thr697). 5. It is concluded that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the MYPT1. These results suggest that flavone causes endothelium-independent relaxation through, at least in part, the inhibition of p160 Rho-associated coiled-coil-containing protein kinase (ROCK) signalling.     

REGIONAL DIFFERENCES IN THE MODULATORY ROLE OF THE EPITHELIUM IN SHEEP AIRWAY

• 1 The airway epithelium may modulate smooth muscle responsiveness via the release of biologically active substances, such as nitric oxide (NO) and prostaglandins. Based on regional differences in structure and function described for the airway epithelium, we performed a comparative study on the responsiveness of sheep isolated, epithelium‐intact or ‐denuded, first‐ to fourth‐order bronchi to acetylcholine (ACh).
• 2 We performed contractility studies using KCl or cholinergic stimuli in the presence or absence of NO or prostaglandin‐related drugs in epithelium‐intact and epithelium‐denuded bronchial strips obtained from all four airway regions. We also studied the expression of NO synthase (NOS), using the NADPH‐diaphorase staining technique, and the effect of airway epithelium removal on the synthesis of NO metabolites in the different bronchi orders.
• 3 There was no difference in the response of first‐ to fourth‐order epithelium‐intact bronchi to ACh (1 nmol/L–100 mmol/L) or KCl (5–100 mmol/L). Removal of the epithelium had no effect on ACh‐induced contractions of first‐ and second‐order bronchi, but increased responses of third‐ and fourth‐order bronchi to ACh. The NO synthase inhibitor N^G‐nitro‐l‐ arginine methyl ester (100 µmol/L) increased ACh‐induced contractions of fourth‐order epithelium‐intact bronchi only. The NO donor sodium nitroprusside (1 nmol/L–1 mmol/L) equally relaxed 1 µmol/L carbachol‐precontracted epithelium‐denuded first‐ and fourth‐order bronchi.
• 4 Although NAPDH–diaphorase staining demonstrated no regional differences in NOS expression, basal levels of NO metabolites were 4.5‐fold greater in fourth‐ compared with second‐order epithelium‐intact bronchi.
• 5 The cyclo‐oxygenase inhibitor indomethacin (10 µmol/L) had no effect on ACh‐induced contractions of first‐ to fourth‐order epithelium‐intact bronchi, but decreased responses of fourth‐order epithelium‐denuded bronchi to ACh. The contractile effect of the thromboxane A₂ mimetic U‐46619 (1 nmol/L–10 µmol/L) was greater in fourth‐ compared with first‐order epithelium‐denuded bronchi.
• 6 In conclusion, the sheep airway epithelium exhibits regional differences in its modulatory role and this is particularly apparent in small bronchi.

     

7‐difluoromethyl‐5,4′‐dimethoxygenistein,a novel agent protecting against vascular endothelial injury caused by oxidative stress

1. Genistein is known to protect the vascular endothelium. However, genistein exhibits poor bioavailability, which limits its use in the treatment of cardiovascular diseases. 7‐Difluoromethyl‐5,4′‐dimethoxygenistein (dFMGEN), prepared by the difluoromethylation and alkylation of genistein, is a new active chemical entity. The protective effects of dFMGEN against vascular endothelial injury caused by oxidative stress were investigated in the present study. 2. Human umbilical vein endothelial cells were treated with either genistein (10 μmol/L) or various concentrations of dFMGEN (0.1, 0.3, 1, 3 and 10 μmol/L) for 30 min before exposure to 1 mmol/L H₂O₂ for 24 h. The generation of reactive oxygen species (ROS) was assessed by fluorescence flow cytometry, the release of lactate dehydrogenase (LDH) was examined by biochemical assay, cell viability was measured by the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide assay, cell apoptosis was detected by flow cytometry and the expression of caspase 3 was examined by western blot analysis. 3. Pretreatment with 0.1, 0.3, 1, 3 and 10 μmol/L dFMGEN decreased the generation of ROS and the release of LDH in H₂O₂‐exposed vascular endothelial cells, enhanced cell viability in a concentration‐dependent manner over the concentration range 0.1–10 μmol/L, suppressed H₂O₂‐induced apoptosis of vascular endothelial cells and downregulated the expression of caspase 3. The protective effect of 10 μmol/L dFMGEN against oxidative stress‐induced endothelial injury was stronger than that of 10 μmol/L genistein. 4. The results of the present study suggest that dFMGEN can protect against vascular endothelial injury caused by oxidative stress.     

A role for Rho-kinase in Ca-independent contractions induced by phorbol-12,13-dibutyrate

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.     

Heat shock protein 90 mediates cytoprotection by H₂S against chemical hypoxia-induced injury in PC12 cells

1. Increasing evidence indicates that hydrogen sulphide (H₂S) may serve as an important biological cytoprotective agent. Heat shock protein (Hsp) 90 can attenuate stress‐induced injury. However, whether Hsp90 mediates the cytoprotective effect of H₂S against chemical hypoxia‐induced injury in PC12 cells is not known. 2. In the present study, CoCl₂ (a chemical hypoxia mimetic) was used to treat PC12 cells to create a model of chemical hypoxia. To explore the role of Hsp90 in the cytoprotection afforded by H₂S against chemical hypoxia‐induced injury, 2 μmol/L 17‐allylaminogeldanamycin (17‐AAG), a selective inhibitor of Hsp90, was administered for 30 min prior to preconditioning with 400 μmol/L NaHS, followed by chemical hypoxia. 3. Cobalt chloride reduced cell viability (by 52.7 ± 1.5%), increased PC12 cell apoptosis (by 42.1 ± 1.5%), induced reactive oxygen species (ROS) by 3.79% compared with control and induced the dissipation of mitochondrial membrane potential (MMP) by 2.56% compared with control. 4. Pretreatment of PC12 cells with 100–400 μmol/L sodium hydrosulphide (NaHS), an H₂S donor, for 3 h prior to exposure to 600 μmol/L CoCl₂ provided significant, concentration‐dependant protection to PC12 cells against CoCl₂‐induced cytotoxicity. Specifically, pretreatment of PC12 cells with 400 μmol/L NaHS decreased apoptosis to 16.77 ± 1.77% and blocked the CoCl₂‐induced increase in ROS production and loss of MMP. 5. At 400 μmol/L, NaHS upregulated Hsp90 in a time‐dependant manner (over the period 0–180 min). In addition to its effects on Hsp90 expression, NaHS pretreatment of PC12 cells augmented the overexpression of Hsp90 induced by 600 μmol/L CoCl₂ by 1.38‐fold (P < 0.01). 6. Treatment of PC12 cells with 2 μmol/L 17‐AAG for 30 min prior to NaHS pretreatment blocked the overexpression of Hsp90 induced by NaHS preconditioning, as evidenced by decreased cell viability (by 54.2 + 1.2%; P < 0.01), increased PC12 cell apoptosis (by 36.6 ± 1.2%; P < 0.01) and increasing ROS production. 7. The findings of the present study provide novel evidence that Hsp90 mediates H₂S‐induced neuroprotection against chemical hypoxia‐induced injury via anti‐oxidant and anti‐apoptotic effects.     

Warifteine,a bisbenzylisoquinoline alkaloid,induces relaxation by activating potassium channels in vascular myocytes

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Analysis of Adenosine Vascular Effect in Isolated Rat Aorta: Possible Role of Na+/K+‐ATPase

Abstract: The present experiments were undertaken in order to examine the effect of adenosine in isolated rat aorta, to investigate the possible role of intact endothelium and endothelial relaxing factors in this action and to determine which population of adenosine receptors is involved in rat aorta response to adenosine. Adenosine (0.1–300 μM) produced concentration‐dependent (intact rings: pD₂=4.39±0.09) and endothelium‐independent (denuded rings: pD₂=4.52±0.12) relaxation of isolated rat aorta. In the presence of high concentration of K⁺ (100 mM) adenosine‐evoked relaxation was significantly reduced (maximal relaxation in denuded rings: control – 92.1±9.8 versus K⁺– 54.4±5.0). Similar results were obtained after incubation of ouabain (100 μM) or glibenclamide (1 μM). In K⁺‐free solution, K⁺ (1–10 mM)‐induced rat aorta relaxant response was significantly inhibited by ouabain (100 μM). Application of indomethacin (10 μM), N^G‐nitro‐L‐arginine (10 μM) or tetraethylammonium (500 μM) did not alter the adenosine‐elicited effect in rat aorta. 8‐(3‐Chlorostyril)‐caffeine (0.3–3 μM), a selective A_2A‐receptor antagonist, significantly reduced adenosine‐induced relaxation of rat aorta in a concentration‐dependent manner (pK_B=6.57). Conversely, 1,3‐dipropyl‐8‐cyclopentylxanthine (10 nM), an A₁‐receptor antagonist, did not affect adenosine‐evoked dilatation. These results indicate that in isolated rat aorta, adenosine produces endothelium‐independent relaxation, which is most probably dependent upon activation of smooth muscle Na⁺/K⁺‐ATPase, and opening of ATP‐sensitive K⁺ channels, to a smaller extent. According to receptor analysis, vasorelaxant action of adenosine in rat aorta is partly induced by activation of smooth muscle adenosine A_2A receptors.