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The present study investigated the influence of gender on the changes underlying endothelial dysfunction in hyperlipidemia during aging. Isometric tension in rings (with endothelium) of the aortae and superior mesenteric arteries from apolipoprotein-E deficient mice was determined in wire myographs. Nitric oxide (NO)- and endothelium-dependent hyperpolarization (EDH)-mediated relaxations were smaller in the aortae and mesenteric arteries of 32weeks old males than eight weeks old males. In females, NO- and EDH-mediated relaxations were impaired only at 84weeks of age. The levels of reactive oxygen species were elevated in the blood vessels of 32weeks old males, but not females. Acute in vitro treatment with 17β-estradiol and apocynin improved NO- and EDH-mediated relaxations in 32weeks old males but not in 84weeks old males. Relaxations to SKA-31, activator of intermediate (IKCa) and small (SKCa) conductance calcium-activated potassium channels, were attenuated in the mesenteric arteries of 32weeks old males. Such impairment was restored by acute treatment with apocynin. These findings suggest that male hyperlipidemic mice develop endothelial dysfunction at an earlier age than females. This endothelial dysfunction is associated with impaired NO bioavailability and reduced IKCa and SKCa activity. Apocynin and 17β-estradiol restore the endothelial function only in younger male animals but not in older male or female animals.

FigureS1 Effect of (A) NOS inhibitor or (B) GC inhibitor on bradykinin-induced relaxations. Rings from porcine coronary arteries were incubated with either L-NAME (10−4M) or ODQ (10−5M) for 30min. They were then contracted with U46619 (3 × 10−8M) before bradykinin was cumulatively added. n = 6–7. *P < 0.05, significantly different from the control group. FigureS2 Effect of (A) intermediate- plus small-conductance calcium-activated potassium channels inhibitors or (B) large-conductance calcium-activated potassium channel inhibitor on bradykinin-induced relaxations. Rings from porcine coronary arteries were incubated with TRAM-34 (10−6M) plus UCL 1684 (10−6M) or iberiotoxin (10−7M) for 30min. They were then contracted with U46619 (3 × 10−8M) before bradykinin was cumulatively added. n = 7–8. *P < 0.05, significantly different from the control group. FigureS3 Effect of (A) large and intermediate plus small-conductance calcium-activated potassium channels inhibitors and (B) non-selective calcium-activated potassium channel inhibitor on bradykinin-induced relaxations in the absence or presence of kaempferol. Rings from porcine coronary arteries were incubated with charybdotoxin (10−7M) plus apamin (10−6M), TEA (10−3M), with or without kaempferol (3 × 10−6M), for 30min. They were then contracted with U46619 (3 × 10−8M) before bradykinin was cumulatively added. n = 7–8. *P < 0.05, significantly different from the control group. FigureS4 Effect of the combination of NOS and intermediate plus small-conductance calcium-activated potassium channels inhibitors on bradykinin-induced relaxations in the absence or presence of kaempferol. Rings from porcine coronary arteries were incubated with L-NAME (10−4M), TRAM-34 (10−6M) plus UCL 1684 (10−6M) and/or kaempferol (3 × 10−6M), for 30min. They were then contracted with U46619 (3 × 10−8M) before bradykinin was cumulatively added. n = 7–8. *P < 0.05, significantly different from the control group. FigureS5 Effect of large-conductance calcium-activated potassium channel inhibitor on sodium nitroprusside-induced relaxations. They from porcine coronary arteries were incubated with or without iberiotoxin (10−7M) for 30min. They were then contracted with U46619 (3 × 10−8M) before sodium nitroprusside was cumulatively added. n = 6–7. TableS1 Effects of acute treatment with different flavonoids on the contraction to U46619 (3 × 10−8M) before relaxations to bradykinin (10−11 to 10−6M) in porcine coronary arteries. TableS2 Effects of different pharmacological treatments, in the absence or presence of kaempferol (3 × 10−6M), on the contraction to U46619 (3 × 10−8M) before relaxations to bradykinin (10−11 to 10−6M) in porcine coronary arteries. TableS3 Effects of different potassium channel inhibitor, in the absence or presence of kaempferol (3 × 10−6M), on the EC50 and Emax values of concentration–relaxation curves of bradykinin (10−11 to 10−6M) in porcine coronary arteries contracted by U46619 (3 × 10−8M). TableS4 Effects of large-conductance calcium-activated potassium channel inhibitor, in the absence or presence of kaempferol (3 × 10−6M), on the contraction to U46619 (3 × 10−8M) before relaxations to sodium nitroprusside (10−9 to 10−4M) in porcine coronary arteries. Click here to view.(494K, pdf)

Background The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. l-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of l-arginine in modulating the overall vascular response to dexmedetomidine. Methods Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 μM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), l-arginine, (S)-(2-boronethyl)-l-cysteine hydrochloride (arginase inhibitor), NG-hydroxy-l-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed. Results Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with l-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous l-arginine augmented the dexmedetomidine-induced contractions in the presence of l-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-l-cysteine hydrochloride (Emax 16 ± 4%) and NG-hydroxy-l-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as l-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by l-arginine treatment in the presence of l-NAME (N = 4). Conclusions These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of dexmedetomidine by l-arginine depends on arginase activity and the production of urea and ornithine.

Heme oxygenase (HO) converts heme to carbon monoxide, bilirubin, and free iron. The present study investigated whether or not HO-1 induction improves vascular relaxations attributable to endothelium-dependent hyperpolarization (EDH). Thirty-six-week-old spontaneously hypertensive rats were treated with the HO-1 inducer hemin, the HO inhibitor zinc protoporphyrin IX (II) (ZnPP), the antioxidant apocynin, or combinations of these compounds. Isolated mesenteric arteries were prepared for measurement of isometric tension, protein presence, and production of reactive oxygen species (ROS). Hemin potentiated acetylcholine-evoked EDH-type relaxations in the presence of Nω-nitro-l-arginine methyl ester (l-NAME) and indomethacin, while the combined treatment with ZnPP plus hemin prevented these improvements. The intermediate conductance Ca2+-activated K+ channel (IKCa) blocker TRAM-34 and the Na+-K+-ATPase blocker ouabain significantly impaired these hemin-potentiated relaxations. NS309-induced TRAM-34- and ouabain-sensitive relaxations were enhanced by hemin. K+-induced ouabain-sensitive relaxations and the expression of Na+-K+-ATPase were increased by hemin. Thus HO-1 induction improves EDH-type relaxations by augmented activation of IKCa and the downstream Na+-K+-ATPase. Treatment with apocynin showed a similar effect as hemin in impairing ROS production, enhancing K+-induced relaxations, and increasing Na+-K+-ATPase expression, without affecting the expression of HO-1. The effects of hemin and apocynin were not additive. These observations suggest that the effect of HO-1 induction on EDH-type relaxations is possibly due to its antioxidant properties. In vitro treatment with bilirubin, but not carbon monoxide, enhanced EDH-type relaxations and K+-induced ouabain-sensitive relaxations, suggesting that the production of bilirubin may be also involved. The present findings reveal that HO-1 may be a potential vascular-specific therapeutic strategy for endothelial dysfunction in hypertension.

Experiments were designed to determine if Wy14643 ([[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]-acetic acid), a preferential agonist at peroxisome proliferator-activated receptors (PPAR) α , improves vascular function in hypertension, and if so, the mechanism(s) involved. Isometric tension was measured in isolated thoracic aorta of spontaneously hypertensive rats (SHR). Wy14643-induced relaxations in SHR aortic rings were greater than those induced by fenofibrate or rosiglitazone (PPARα or PPAR γ agonists, respectively) and were larger in rings with endothelium than those without. Both MK886 [(1-[(4-chlorophenyl)methyl]-3-1,1-dimethylethyl)thio]-( α , α -dimethyl-5-1-methylethyl)-1H-indole-2-propanoic acid (PPAR α antagonist) and GW9662 (2-chloro-5-nitrobenzanilide) (PPAR γ antagonists) inhibited Wy14643-induced relaxations. The inhibitory effect of MK886 was more pronounced in rings with endothelium than those without. In SHR aortic rings with endothelium, L-NAME ( N ω -nitro-L-arginine methyl ester, nitric oxide synthase inhibitor), ODQ (1H-1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one, soluble guanylyl cyclase inhibitor) and compound C [adenosine monophosphate-activated protein kinase (AMPK) inhibitor] reduced Wy14643-induced relaxations. Western blotting studies indicated that Wy14643 and fenofibrate, but not rosiglitazone, increased the phosphorylation of endothelial nitric oxide synthase and AMPK; these effects were abolished by compound C but not L-NAME. Endothelium-dependent contractions evoked by acetylcholine in quiescent SHR aorta in the presence of L-NAME were reduced by Wy14643 and fenofibrate but not by rosiglitazone. MK886, but not GW9662, prevented this effect. Wy14643 and fenofibrate inhibited acetylcholine-induced prostanoid release to the same extent. These findings suggest that PPARα agonists induce nitric oxide-mediated relaxation through activation of AMPK and reduce the release of endothelium-dependent contracting factors. Because also of the ability to activate smooth muscle PPARγ to induce relaxation, Wy14643 offers additional protection against vascular dysfunction of spontaneous hypertension.

Genistein, a phytoestrogen present in soybeans, has well established vasodilator properties. The present study examined the mechanisms involved in the rapid vascular effects of genistein. Endothelium-dependent relaxations and contractions, induced by acetylcholine and the calcium ionophore A23187, were obtained in isolated aortic rings from male spontaneously hypertensive rats (SHR). Acute exposure to genistein potentiated relaxations and reduced contractions induced by the two agonists. Both effects of genistein were not affected by transcription- and translation-inhibitors or by tyrosine kinase inhibition. The potentiation of acetylcholine and A23187-induced relaxation by genistein was inhibited by NF023 and GP antagonist-2A, selective G(i) and G(q) α-subunit antagonists, respectively, but not by NF449, a selective G(s) α-subunit antagonist. These G protein antagonists did not alter the inhibitory effect of genistein on acetylcholine and A23187-induced contractions. The potentiation of A23187-induced relaxations by genistein was not inhibited by the conventional estrogen receptor (ER) antagonist, ICI 182,780, but inhibited by the specific ER-α antagonist, MPP, and by the epidermal growth factor receptor (EGFR) inhibitor, AG1478. It was mimicked by heparin-binding epidermal growth factor (HB-EGF). Activation of EGFR and endothelial nitric oxide synthase (eNOS) was detected in genistein-treated rings using Western blotting. These data suggest that the rapid vascular actions of genistein are mediated by non-genomic pathways and are unrelated to its tyrosine kinase inhibitory properties. Furthermore, genistein transactivates EGFR through membrane ERα via G protein-coupled pathways. This in turn enhances eNOS phosphorylation and hence endothelial function in the aorta of the SHR.

Dexmedetomidine is an α(2)-adrenoceptor agonist and anesthetic. The present study was designed to characterize the receptor subtypes and the downstream mechanisms of the vascular effects of dexmedetomidine in small (mesenteric artery) and large (aorta) arteries ex vivo. Isometric tension was measured in Sprague-Dawley rat mesenteric and aortic rings (with or without endothelium). To study relaxations, cumulative concentrations of dexmedetomidine, 5-bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine, (UK14304), or clonidine were added to rings contracted with 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F(2α) (U46619) in the presence or absence of indomethacin; N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME); 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate (BRL44408); 2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239); l-657,743, (2S-trans)-1,3,4,5',6,6',7,12b-octahydro-1',3'-dimethyl-spiro[2H-benzofuro[2,3-a]quinolizine-2,4'(1'H)-pyrimidin]-2'(3'H)-one hydrochloride hydrate (MK912); rauwolscine; prazosin; or pertussis toxin. To study contractions, dexmedetomidine was added to quiescent rings without endothelium or after incubation with l-NAME, rauwolscine, prazosin, indomethacin, or 3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronaphth)-1-yl]propionic acid (S18886). Dexmedetomidine evoked relaxation at low concentrations (10 pM-30 nM) followed by contraction at higher concentrations (>30 nM) in the mesenteric artery. In the aorta, the relaxation was significantly smaller. The relaxation to dexmedetomidine depended on nitric oxide, endothelium, and G(i) protein, and it was mediated by α(2A/D)-adrenoceptors and possibly α(2B)-adrenoceptors. The contraction was mediated mainly by α(2B)- and α(1)-adrenoceptors and involved the action of prostanoids. UK14304 and clonidine induced greater and smaller relaxations, respectively, than dexmedetomidine. In conclusion, depending on the concentration used and the presence of functional endothelium, dexmedetomidine may evoke both relaxation and contraction in isolated arteries. The vascular effects also vary depending on the blood vessel studied. Its vascular effect is different from that of clonidine and UK14304.

Acute inhibition of nitric-oxide synthase (NOS) unmasks the release of endothelium-derived contracting factors (EDCFs). The present study investigated whether chronic inhibition of NOS modulates endothelium-dependent contractions. Eighteen-week-old male Sprague-Dawley rats were treated by daily gavage for 6 weeks with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (60 mg/kg) or vehicle (distilled water; 1 ml/kg). Chronic treatment with L-NAME increased arterial blood pressure. Isometric tension was measured in aortic rings with or without endothelium. Endothelium-dependent relaxations to acetylcholine and the calcium ionophore 5-(methylamino)-2-[(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187) were reduced in preparations from L-NAME-treated rats. The reduction in relaxation to A23187 was partially reversed by L-arginine (1 mM). In quiescent aortic rings, A23187 caused contractions in the presence of L-NAME and intact endothelium. The A23187-induced contractions were greater in rings from the L-NAME-treated rats than in those from the control group. These contractions were abolished by the cyclooxygenase (COX)-2 inhibitor N-[2-cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS-398) and the thromboxane-prostanoid (TP) receptor antagonist 3-((6R)-6-{[(4-chlorophenyl)sulfonyl]amido}-2-methyl-5,6,7,8-tetrahydronaphthalen-1-yl)propanoate (S18886), but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560). Chronic L-NAME treatment reduced the level of nitric oxide in the plasma but increased COX activity in the aortic rings. Western blotting and immunohistochemical staining showed that endothelial NOS expression was reduced in the aortae of the chronic L-NAME-treated group. COX-1 expression was augmented slightly, whereas COX-2 expression was up-regulated markedly. The TP receptor expression was comparable with control. These experiments demonstrate that chronic NOS inhibition increases endothelium-dependent contractions of the rat aorta by inducing COX-2 expression and augmenting the production of EDCF.

Phospholipase A2(PLA2), a regulatory enzyme found in most mammalian cells, catalyzes the breakdown of membrane phospholipids to arachidonic acid. There are two major cytosolic types of the enzyme, calcium-dependent (cPLA2) and calcium-independent (iPLA2) PLA2. The present study investigated whether or not iPLA2plays a role in the endothelium-dependent contractions of the aorta of the spontaneously hypertensive rat and its normotensive counterpart, the Wistar-Kyoto rat. The presence of iPLA2in the endothelial cells was identified by using immunochemistry and immunoblotting. Aortic rings with and without the endothelium were suspended in organ chambers for isometric tension recording. The production of prostanoids was measured by using enzyme immunoassay kits. iPLA2was densely distributed in endothelial cells of the aorta of both strains. At 3 × 10−6M, the selective iPLA2inhibitor, bromoenol lactone (BEL), abrogated endothelium-dependent contractions induced by acetylcholine but not those evoked by the calcium ionophore A-23187. The effects of BEL were similar in the aortae of Wistar-Kyoto and spontaneously hypertensive rats. The nonselective PLA2inhibitor quinacrine abolished the contractions triggered by both acetylcholine and A-23187, whereas the store-operated calcium channel inhibitor SKF-96365 prevented only the acetylcholine-induced contraction. The acetylcholine- but not the A-23187-induced release of 6-keto prostaglandin F1αwas inhibited by BEL. The release of thromboxane B2by either acetylcholine or A-23187 was not affected by BEL. In conclusion, iPLA2plays a substantial role in the generation of endothelium-derived contracting factor evoked by acetylcholine.

SummaryBackgroundEpigallocatechin-3-gallate (EGCG), which has been shown to have potent antioxidant effect, comprises 80% of catechins in Chinese green tea. This study was to investigate whether cigarette smoke (CS) exposure would induce lung morphological changes and oxidative stress in the CS-exposed rat model, and whether Chinese green tea (Lung Chen tea with EGCG as its main active ingredient) consumption would alter oxidative stress in sera and lung leading to protection of CS-induced lung damage.MethodsSprague-Dawley rats were randomly divided into four groups, i.e. sham air (SA), 4% CS, 2% Lung Chen tea plus SA or 4% CS. Exposure to SA or 4% CS was performed for 1h/day for 56 days in ventilated smoking chambers. Sera and lung tissues were collected 24h after last CS exposure for histology and all biochemical assays.ResultsAirspace enlargement and goblet cell hyperplasia were observed after 56-day CS exposure alone, which were abolished in the presence of green tea consumption. Serum 8-isoprostane level was significantly elevated (p

Rho kinase is involved in the pathogenesis of hypertension, which favors the occurrence of endothelium-dependent contractions. The present study was designed to determine the effects of two Rho kinase inhibitors, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine (fasudil)] and Y27632 [(+)-( R )- trans -4-(1-aminoethyl)- N -(4-pyridyl) cyclohexane carboxamide dihydrochloride], on endothelium-dependent and -independent contractions. Isometric tension of 1-year-old spontaneously hypertensive rat and Wistar Kyoto aortae were measured. In the presence of N ω-nitro-l-arginine methyl ester, HA1077, and Y27632 reduced endothelium-dependent contractions caused by acetylcholine and the calcium ionophore 5-(methylamino)-2-[[2 R ,3 R ,6 S ,8 S ,9 R ,11 R )-3,9,11-trimethyl-8-[(1 S )-1-methyl-2-oxo-2-(1 H -pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187). The Rho kinase inhibitors did not significantly affect prostacyclin production measured as 6-keto prostaglandin F1α. They nearly abolished endothelium-independent contractions to (5 Z )-7-[(1 R ,4 S ,5 S ,6 R )-6-[(1 E ,3 S )-3-hydroxy-1-octenyl]-2-oxabicyclo-[2.2.1]hept-5-yl]-5-heptenoic acid (U46619), prostaglandin F2α, and phenylephrine. Western blotting revealed a comparable expression of Rho kinase in the aortae of the two strains. The reduction by Rho kinase inhibitors of endothelium-dependent contractions is mainly because of their direct effect on the vascular smooth muscle cells.

Aim: Endothelium-dependent relaxations to certain neurohumoral substances are mediated by pertussis toxin-sensitive Gi/o protein. Our experiments were designed to determine the role, if any, of pertussis toxin-sensitive G-proteins in relaxations attributed to endothelium-derived hyperpolarizing factor (EDHF). Methods: Pig coronary arterial rings with endothelia were suspended in organ chambers filled with Krebs-Ringer bicarbonate solution maintained at 37 °C and continuously aerated with 95%O 2 and 5% CO 2 . Isometric tension was measured during contractions to prostaglandin F 2α in the presence of indomethacin and N ω -nitro- L -arginine methyl ester ( L -NAME). Results: Thrombin, the thrombin receptor-activating peptide SFLLRN, bradykinin, substance P, and calcimycin produced dose-dependent relaxations. These relaxations were not inhibited by prior incubation with pertussis toxin, but were abolished upon the addition of charybdotoxin plus apamin. Relaxations to the α2-adrenergic agonist UK14304 and those to serotonin were abolished in the presence of indomethacin and L -NAME. Conclusion: Unlike nitric oxide-mediated relaxations, EDHF-mediated relaxations of pig coronary arteries do not involve pertussis toxin-sensitive pathways and are Gi/o protein independent.

Background and purpose: Experiments were designed to determine the modulation by nitric oxide (NO) and endothelium-dependent hyperpolarizations (EDHF-mediated responses) of endothelium-dependent contractions in renal arteries of normotensive and hypertensive rats. Experimental approach: Rings, with or without endothelium, of renal arteries of 8-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were suspended in myographs for isometric force recording. Key results: ACh evoked relaxations in preparations contracted with phenylephrine. L-NAME (inhibitor of NOS) attenuated (WKY) or abolished (SHR) these relaxations. TRAM-34 plus UCL 1684 (inhibitors of EDHF-mediated responses) did not decrease the relaxation, except in rings of WKY when L-NAME was also present. High concentrations of ACh caused a secondary increase in tension, augmented in rings of WKY by L-NAME or TRAM-34 plus UCL 1684. The increase in tension was prevented by indomethacin. Under baseline tension, ACh induced endothelium-dependent contractions, prevented by indomethacin (COX inhibitor) or terutroban (TP receptor antagonist). The calculated endothelium-dependent contractions were larger in rings of SHR compared with those of WKY. In preparations of SHR, the contractions were augmented by L-NAME in the presence of SC19220 (EP-1 receptor antagonist). In arteries of WKY, the endothelium-dependent contractions were augmented by TRAM-34 plus UCL 1684. The responses were reduced by SC19220. Conclusions and implications: In the renal artery of the rat, EDCF-mediated contractions are augmented by hypertension. The endothelium-dependent contractions are facilitated by NOS inhibition (in the presence of an EP-1 receptor antagonist) and by the withdrawal of EDHF-mediated responses. British Journal of Pharmacology (2008) 155, 217–226; doi:10.1038/bjp.2008.256; published online 23 June 2008

Soy consumption is associated with a lower risk of atherosclerotic disease in the oriental population. Genistein is a soy isoflavone bearing estrogenic properties. Previous experiments in our laboratory demonstrated the potentiation of endothelium-independent relaxation of coronary artery by both estrogen and genistein. The potentiating effects of both estrogen and genistein were mediated through the cAMP-signaling pathway. We hypothesize that genistein could enhance protein kinase A (PKA) activity in porcine coronary artery smooth muscle, thereby offering a mechanism for the potentiation of vascular relaxation by genistein. In our study, a high concentration of genistein (10(-4.5) M) significantly increased PKA activity in porcine coronary artery rings. While genistein at 10(-5.5) M and forskolin at 10(-7) M had no effect on PKA activity, the combination of the two compounds at the prescribed concentrations caused a significant increase in PKA activity. The increase in PKA activity by genistein was abolished by SQ 22536 (adenylate cyclase blocker), but not by NF 449 (Gs protein blocker) or ICI 182780 (estrogen receptor antagonist). Our results suggest that the action of genistein is mediated via adenylate cyclase, but does not appear to involve Gs protein or ICI 182780-sensitive estrogen receptor.

Background and Purpose: Experiments were designed to assess whether or not the intracellular concentration of calcium and reactive oxygen species (ROS) increase in endothelial cells of the rat thoracic aorta in response to releasers of endothelium-derived contracting factor (EDCF) and if so, whether or not a difference exists between spontaneously hypertensive (SHR) and normotensive (WKY) rats. Experimental approach: Calcium and ROS were measured by confocal microscopy, using Fura-red in combination with Fluo-4 and dichlorodihydrofluorescein diacetate, respectively. Key results: Acetylcholine caused a rapid increase in cytosolic calcium concentration in endothelial cells of both SHR and WKY, which was significantly more pronounced in aortae of the former strain. This rise of calcium was not affected by indomethacin (an inhibitor of cyclooxygenase) or Tiron plus diethyldithiocarbamate acid (DETCA) (membrane permeable antioxidants). In the presence of a nitric oxide synthase blocker, acetylcholine also caused a rapid increase in ROS in endothelial cells of SHR but not in those of WKY. The burst of ROS was prevented by indomethacin or Tiron plus DETCA. Conclusions and implications: These experiments show that endothelial cells of SHR are more prone to calcium and ROS overload upon stimulation with acetylcholine. The abnormal accumulation of calcium is a prerequisite to initiate the release of EDCF and can be mimicked using the calcium ionophore A23187. The sequence of events occurring during endothelium-dependent contractions firstly requires the accumulation of calcium, which then activates cyclooxygenase and produces ROS along with EDCF that in turn stimulates TP-receptors, resulting in EDCF-mediated contractions. British Journal of Pharmacology (2007) 151, 15–23. doi:10.1038/sj.bjp.0707190

Background and purpose: To study the importance of endothelium-derived contracting factors (EDCFs) in arteries of rats with type I diabetes. Experimental approach: Rat femoral arteries were collected four or twelve weeks after induction of diabetes with streptozotocin. Rings, with or without endothelium, were suspended in organ chambers for isometric tension measurement. COX protein levels were determined by Western blotting. Key results: Four weeks after the injection of streptozotocin, the endothelium-dependent relaxations (during contractions to phenylephrine) to A23817 were attenuated, but the endothelium-dependent contractions (quiescent preparations) to the ionophore were augmented. Indomethacin and S18886 prevented the endothelium-dependent contractions, while dazoxiben reduced them in rings from streptozotocin-treated rats, suggesting that thromboxane A2, activating TP- receptors, is involved. Twelve weeks after the injection of streptozotocin, the changes in endothelium-dependent relaxations and contractions to A23187 were even more noticeable. The protein expression of COX-1 was increased in femoral arteries of the diabetic rats. Valeryl salicylate and SC560 inhibited the contractions, suggesting that the EDCFs are produced by COX-1. At that time, a combination of S18886 with EP1-blockers was required to abolish the contractions, suggesting that the EDCFs involved act at both TP- and EP-receptors. Rings without endothelium from streptozotocin-treated rats exhibited a reduced maximal contraction to potassium chloride and U46619, combined with hyper-responsiveness to the latter, suggesting that more prolonged diabetes also alters the responsiveness of vascular smooth muscle. Conclusion and Implications: The production of EDCFs is progressively increased in the course of type I diabetes. Eventually, the disease also damages vascular smooth muscle. British Journal of Pharmacology (2007) 150, 624–632. doi:10.1038/sj.bjp.0706999

This study investigated the response of the human umbilical artery to various agonists in order to determine which agents might be involved in umbilical cord closure. Umbilical artery rings from 22 cesarean deliveries were suspended in tissue baths containing Krebs-Henseleit solution under low and normal oxygen conditions. Samples were challenged with 5-hy-droxytryptamine, acetylcholine, angiotensin II, bradykinin, endothelin-1, histamine, norepinephrine, phenylephrine, and U46619 (a thromboxane A2 mimetic). Only 5-hydroxytryptamine, bradykinin, endothelin-1, histamine, and V46619 produced appreciable maximal contractions. Furthermore, although the contractile force generated by these agonists was diminished in normal oxygen conditions, this reduction affected responses to 5-hydroxytryptamine and U46619 least. 5-Hydroxytryptamine and U46619 demonstrated the characteristics most likely to be associated with closure of the umbilical artery, ie, they elicited strong, sustained contractions at physiologic concentrations and under both normal and low-oxygen conditions. These data suggest that closure of the umbilical artery may be mediated by 5-hydroxytryptamine and thromboxane A2.

Current consensus suggests that lysophosphatidylcholine is the major detrimental factor in oxidized low-density lipoprotein that may contribute to the alterations of vasomotor responses associated with atherosclerosis. This study investigated the influences of lysophosphatidylcholine and major lipid components in oxidized low-density lipoprotein on vascular relaxation. We also determine if there was any interaction between these phospholipid components on relaxation. Porcine coronary artery rings were incubated with lysophosphatidylcholine, phosphatidylcholine or sphingomyelin. After contraction by the thromboxane A2 mimetic U46619, rings were relaxed with bradykinin and calcium ionophore A23187. Lysophosphatidylcholine with a higher proportion of stearoyl-lysophosphatidylcholine to palmitoyl-lysophosphatidylcholine ratio caused greater reduction of relaxational responses. While phosphatidylcholine and sphingomyelin had no effect on vascular relaxation, they reduced the ability of lysophosphatidylcholine to impair vascular relaxation. Our results thus suggested that the effectiveness of oxidized low-density lipoprotein at inhibiting vasodilatory responses may be determined by the relative proportion of different types of lysophosphatidylcholine as well as the amount of other phospholipid components: phosphatidylcholine and sphingomyelin.

Both endothelin-1 (ET-1) and platelet-activating factor (PAF) have been suggested to play a role in the regulation of the cardiovascular system. In view of the limited data regarding the interaction between ET-1 and PAF, the hemodynamic effects of ET-1 and PAF, either alone or in combination, were investigated in the current study. Anesthetized male Sprague Dawley rats received bolus intravenous injections of ET-1 (1 and 2 nmol/kg) and/or PAF (0.075, 0.15 and 0.3 nmol/kg). In some experiments, the ET receptor antagonist, FR-139317 (2.5 or 5 mg/kg), were injected 5 min before the administration of ET-1 or PAF. ET-1 caused a biphasic response consisting of an initial depressor followed by a delayed but sustained pressor response. Injection of PAF to anesthetized rats resulted only in a decrease in arterial blood pressure. Interestingly, the pressor effect of ET-1 was significantly enhanced in the concomitant presence of PAF. Pretreatment with FR-139317 inhibited the magnitude of ET-1-induced hypertension and increased the duration of the depressor action of ET-1. The time-course of PAF-induced decrease of arterial blood pressure was also prolonged in rats pretreated with FR-139317. These data therefore suggested that ET receptors were activated, either directly or indirectly, by PAF, possibly to facilitate the return of blood pressure to resting level following a depressor response. Thus the activation of ET receptors by PAF might result in the enhancement of the pressor response of ET-1 observed in the current study.

Peroxisome proliferator-activated receptor-α agonists, which are used clinically as lipid-lowering drugs, acutely reduce H2O2-induced contractions in aortae of hypertensive rats by inhibiting the a...

Hyperlipidemia is a well-known risk factor for atherosclerosis and statins are widely used to treat patients with elevated levels of lipids in their plasma. Notwithstanding the proven benefits of statin drugs on both primary and secondary prevention of heart disease, the high cost of statin treatment, in addition to possible side effects such as liver function abnormalities, may limit their widespread use. We conducted a study on a natural product as an alternative to statin treatment. Cholestin, a dietary supplement, is prepared from rice fermented with red yeast (Monascus purpureus), which has been shown to significantly decrease total cholesterol levels in hyperlipidemic subjects. Our objective was to determine the cellular effect of Cholestin on cholesterol synthesis in human hepatic cells (HepG2) and the mechanism by which it caused a change in lipid metabolism. Cholestin had a direct inhibitory effect on HMG-CoA reductase activity (78-69% of control). Cholesterol levels in HepG2 cells treated with Cholestin (25-100 microg/mL) were significantly reduced in a dose-dependent manner (81-45% of control, respectively). This reduction was associated with decreased synthesis and secretion of both unesterified cholesterol (54-31 and 33-14% of control, respectively) and cholesteryl ester (18-6 and 37-19% of control, respectively). These results indicate that one of the anti-hyperlipidemic actions of Cholestin is a consequence of an inhibitory effect on cholesterol biosynthesis in hepatic cells and provide the first documentation of a biomolecular action of red yeast rice.

Epidemiological studies have revealed that postmenopausal estrogen replacement therapy results in a marked reduction in the risk for cardiovascular diseases. In the present study, we evaluated plasma lipoprotein profile as well as homocysteine levels in 145 post‐menopausal and premenopausal Chinese women living in Hong Kong. We also investigated the effect of hormone‐replacement therapy (HRT) with estrogen or estrogen combined with progestin on plasma lipoprotein profile and homocysteine concentrations in those individuals. Postmenopausal women displayed significantly higher plasma levels of total cholesterol, LDL‐cholesterol and apoB as well as higher plasma homocysteine levels than that of premenopausal women. HRT with either estrogen (17β-estradiol or conjugated equine estrogen) alone or estrogen combined with progestin for 3.5–4.5 years significantly improved the lipoprotein profile in postmenopausal women by decreasing the levels of total cholesterol (12–20% reduction), LDL-cholesterol (26–29% reduction) and apoB (21–25% reduction). In women treated with 17β‐estradiol or conjugated equine estrogens their plasma levels of apoAI were significantly elevated (18% elevation) as compared to non-users. HRT also reduced plasma concentrations of homocysteine (13–15% reduction). In conclusion, we found that long-term HRT was associated with improvement in plasma lipoprotein profile and a reduction in homocysteine concentration in postmenopausal women. These results support the notion that the improvement of lipoprotein profile and a reduction in homocysteine concentration may contribute to the beneficial effect of HRT on cardiovascular risk.

The present study extends our previous finding that the endothelium-independent relaxation in porcine coronary artery rings is enhanced after short-term (20 min) exposure to a physiological concentration (1 nM) of 17beta-estradiol and demonstrates that this effect may be attributable to activation of the cyclic AMP pathway. Isometric tension was recorded in isolated rings of porcine coronary arteries. Relaxation by levcromakalim and sodium nitroprusside, but not bradykinin and calcium ionophore A23187, were significantly potentiated following 20 min treatment with 1 nM 17beta-estradiol. This enhancing effect was insensitive to the transcriptional and translational inhibitors, actinomycin D and cycloheximide respectively and absent following repeated washing of the rings prior to construction of relaxation-response curves. The potentiating actions of 1 nM 17beta-estradiol on endothelium-independent relaxation were mimicked by the cyclic AMP analogue 8-Bromo-cyclic AMP and the protein kinase A activator Sp-cyclic AMPS but not by the cyclic GMP analogue 8-Bromo-cyclic GMP. The modulatory effect of 17beta-estradiol was increased in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The cyclic AMP-dependent protein kinase A inhibitor Rp-cyclic AMPS, but not the cyclic GMP antagonist Rp-8-Bromo-cyclic GMPS, effectively inhibited the enhancing effects 1 M 17beta-estradiol had on the relaxation responses of levcromakalim and sodium nitroprusside. These data support our earlier findings that physiologically relevant concentrations of 17beta-estradiol can acutely modify vasorelaxation in vitro. Furthermore, we report that this short-term effect of 17beta-estradiol on vasorelaxation appears to be mediated via non-genomic pathways and involves the cyclic AMP cascade.

Objectives: While there are many suggested reasons for the marked gender bias in cardiovascular events, much of the available data indicate that circulating estrogens are cardioprotective. The possibility that endogenous androgens may be detrimental to the cardiovascular system has received relatively less attention. We investigated the short-term modulatory effects of various concentrations of testosterone on vascular function in isolated porcine coronary artery rings. Results: The higher concentrations (>1 μM) of testosterone relaxed U46619-contracted coronary artery rings in an endothelium-independent manner. This direct effect was insensitive to the testosterone receptor antagonists, flutamide and cyproterone acetate. Short-term exposure (20 min) to low levels of testosterone (1–100 nM), which were ineffective on their own on vascular function, significantly diminished relaxation to bradykinin and calcium ionophore A23187 but not those produced by levcromakalim and sodium nitroprusside. The inhibitory effect observed with 1 nM testosterone was only partially reversed by flutamide and cyproterone acetate and unaltered in the presence of actinomycin D and cycloheximide. Conclusions: These results demonstrate that acute treatment with testosterone, at concentrations that have no effect on their own, reduces vasorelaxation. Furthermore, they suggest that this modulatory action may be in part independent of the classical testosterone receptor since it was not completely sensitive to the anti-androgens and was not inhibited by the transcriptional and translational inhibitors. These findings support the postulation that testosterone may have unfavorable influences on vascular function.

The objective of the research project was to investigate whether fenofibrate treatment may alter the biochemical content of the oxidized LDL and consequently its ability to impair the endothelium-dependent relaxation in hyperlipidemic patients. We hypothesized that fenofibrate treatment of hyperlipidemic patients may attenuate the ability of their oxidized LDL to impair the endothelium-dependent relaxation of the blood vessels as a consequence of fenofibrate-induced changes to the content and composition of lysoPC in the LDL molecule.

Inasmuch as the oxidation of low-density lipoprotein (Ox-LDL) may play a key role in the initiation and progression of atherosclerosis, it has become increasingly important to identify potential antioxidants. Panax quinquefolium saponins (PQS) are extracted from the stems and leaves of the North American form of ginseng,Panax quinquefolium. Our previous studies have indicated that PQS (0.25-1 mg/ml) can protect against oxidation of LDLin vitro. The purpose of the current work was to investigate the potential interaction of lower concentrations of PQS (1-100 μg/ml) with vitamin C on the reduction of LDL oxidation. LDL was isolated from the plasma of healthy human donors by sequential ultracentrifugation. Native LDL (0.05 or 0.2 mg/ml) was incubated with PQS and/or vitamin C for 30 min at 20°C. Oxidative modification was initiated with 2 μM or 5 μM CuSO4 at 37°C for 0-24 h. Pretreatment with PQS (100 μg/ml) reduced alterations in phospholipids, lipid peroxide levels and relative electrophoretic mobility of Ox-LDL. The presence of vitamin C (1-10 μM) significantly enhanced the protective effects of PQS. Pretreatment with PQS (1-100 μg/ml) resulted in concentration-dependent inhibition of LDL oxidation and prolongation of lag time as determined from measurements of conjugated lipid hydroperoxide content in Ox-LDL samples. Interestingly, the inhibitory actions of lower amounts of PQS (1 and 10 μg/ml) on the formation of conjugated dienes were significantly increased when vitamin C (0.1 or 1 μM) was present. In conclusion, our results suggest that PQS not only have direct antioxidant property but at low concentrations, their actions can be enhanced by vitamin C.

The progestin element of hormone replacement therapy may reduce the cardioprotective actions of the estrogen component. Only high concentrations (microM) of progesterone directly relaxed U46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F2alpha)-pre-contracted porcine coronary artery rings. A low concentration of progesterone (1 nM), with no effects of its own, shifted the relaxation curves of bradykinin and calcium ionophore A23187 to the right while not affecting those of sodium nitroprusside and levcromakalim. The negative influence that 1 nM progesterone exerted on bradykinin- and A23187-mediated relaxation was diminished when 1 nM 17beta-estradiol was concomitantly added to the bathing medium. Conversely, the potentiating actions of 1 nM 17beta-estradiol on relaxations elicited by sodium nitroprusside and levcromakalim were reduced following simultaneous treatment with the same concentrations of progesterone. These findings represent the first evidence for an acute in vitro vascular effect of progesterone at a physiologically relevant concentration and concur with previous in vivo reports demonstrating that progesterone may diminish the beneficial effects of estrogens.

The effects of chronic 17β-estradiol on endothelium-dependent relaxation to acetylcholine (ACh) and contraction to N G-nitro-l-arginine methyl ester (l-NAME), and endothelium-independent relaxation to sodium nitroprusside (SNP) were examined on blood vessels from rats with chronic heart failure (CHF). Two groups of ovariectomized female (50–60 days) rats were implanted with pellets containing 17β-estradiol (25 µg/day) or vehicle, and given ligation of the left main coronary artery 1 week later. Another group of ovariectomized rats was implanted with vehicle pellets, and sham-operated. After 7 weeks, thoracic aortic rings, pulmonary artery rings, and portal vein strips were prepared for in vitro studies. Relative to sham-operated rats treated with the vehicle, vessels from vehicle-treated, coronary-ligated rats had similar relaxation to ACh and SNP but reduced response to l-NAME that was significant (P

Oxidized low-density lipoprotein (Ox-LDL) is believed to be involved in the pathogenesis of atherosclerosis. Panax quinquefolium saponins (PQS) are extracted from the stems and leaves of the North American form of ginseng, Panax quinquefolium. Earlier studies have suggested that this extract improves the lipid profile of hyperlipidemic rats and has antioxidant properties in cultured rat cardiac myocytes. The aims of the present study were to investigate the potential of PQS in reducing LDL oxidation as well as limiting the ability of Ox-LDL to impair endothelium-dependent relaxation in rat aortic rings. LDL was isolated from the plasma of healthy human donors by sequential ultracentrifugation. Native LDL (0.2 or 0.3 mg ml ) was incubated with PQS (0.25–1 mg ml ) for 30 min at 20 °C. For comparison, vitamin C (50 μM) was added in place of PQS. Oxidative modification was initiated with 5 μM CuSO4 at 37 °C for 0–24 h. In our hands, PQS concentration-dependently reduced lipid peroxide levels as measured by the amount of thiobarbituric acid reactive substances formed. This range of PQS also retarded the alterations in relative electrophoretic mobility of Ox-LDL in a similar manner. Furthermore, measurement of phospholipid fractions content indicated that PQS could reduce the conversion of phosphatidylcholine to lysophosphatidylcholine in Ox-LDL. Functional studies demonstrated that PQS-pretreated Ox-LDL was less potent than untreated Ox-LDL at impairing endothelium-dependent relaxation in rat aortic rings. In conclusion, our results suggest that PQS has antioxidant properties and that reduction of LDL oxidation by PQS may provide a protective effect against the detrimental actions of Ox-LDL.

Homocysteinemia and hypercholesterolemia are important risk factors associated with the occurrence of arteriosclerotic vascular diseases. A positive correlation between plasma levels of homocysteine and cholesterol was found in homocysteinemic patients as well as in experimental animals. In the present study, the effect of homocysteine on the production and secretion of cholesterol in human hepatoma cell line HepG2 cells was investigated. When cells were incubated with 4 mM homocysteine, the amounts of total cholesterol produced as well as the cholesterol secreted by these cells were significantly increased (from 32 +/- 5 to 74 +/- 5 nmol/mg cellular protein). Further biochemical analyses revealed that the increase in cholesterol was resulted from an enhancement in the production and secretion of the unesterified cholesterol with no concomitant change in the level of cholesteryl esters. The activity of intracellular 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was markedly elevated by 131% and 190% after cells were incubated with homocysteine for 24 and 48 h. Homocysteine also stimulated the secretion of apo B100 by HepG2 cells (from 0.84 +/- 0.11 to 1.37 +/- 0.12 micrograms apolipoprotein B/mg cellular protein). Our results demonstrate that homocysteine stimulates the production and secretion of cholesterol and apolipoprotein B100 in HepG2 cells. The increase in the production of cholesterol induced by homocysteine may contribute to the pathogenesis of arteriosclerosis.

Phosphatidylcholine is the major phospholipid in mammalian tissues and the biosynthesis of phosphatidylcholine in H9c2 cells was previously shown to be stimulated by angiotensin II. In this study, we used the potent AT1 receptor antagonist, losartan, to determine if the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis was mediated by AT1 receptors. H9c2 cells were incubated with angiotensin II in the absence or presence of various concentrations of losartan. The cells were then incubated with [methyl-3H]choline for an additional 60 min and the radioactivity incorporated into phosphatidylcholine and its choline-containing metabolites determined. Losartan at concentrations which block AT1 receptors did not effect phosphatidylcholine biosynthesis mediated by angiotensin II. In contrast, higher concentrations of losartan inhibited radioactivity incorporated into phosphatidylcholine and its metabolites and this was due to a losartan-mediated reduction in choline uptake. Kinetic studies revealed that the losartan-mediated inhibition of choline uptake was competitive. High concentrations of losartan caused a translocation of CTP:phosphocholine cytidylyltransferase from the cytosolic (inactive) to the membrane (active) fraction likely as a compensatory mechanism for the losartan-mediated reduction in new phosphatidylcholine biosynthesis. Incubation of cells with PD123319, a potent AT2-receptor antagonist, did not block the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis. The results suggest that angiotensin II stimulates phosphatidylcholine biosynthesis independent of AT1- and AT2-receptor activation and losartan inhibits phosphatidylcholine biosynthesis by reducing choline uptake in H9c2 cells.

Elevated levels of plasma lipoprotein (a) [Lp(a)] have been considered as a strong risk factor for premature cardiovascular diseases. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of plasminogen activators (PA). Increases in PAI-1 levels with or without a reduction in PA levels have been frequently found in coronary artery disease patients. The present paper examined the effects of oxidized Lp(a) on the production of PAI-1 in cultured human umbilical vein endothelial cells (HUVEC). Lp(a) and Lp(a)-free, low density lipoprotein (LDL) were prepared using lysine-Sepharose 4B affinity chromatography. Incubations with 10 −8 M levels of native Lp(a) moderately increased the levels of biologically active PAI-1 in post-culture medium of HUVEC compared to that with equimolar concentrations of native Lp(a)-free LDL. The release of PAI-1 induced by Lp(a) was enhanced by oxidative modification with copper ion. The stimulation of oxidized Lp(a) on PAI-1 production reached plateau in EC treated with 10–20 nM oxidized Lp(a) modified by 5 μ M CuSO 4 . Treatment with 0.2 μ g/ml of actinomycin D significantly reduced native and oxidized Lp(a)-induced PAI-1 overproduction in EC. Increases in the steady state levels of PAI-1 mRNA were detected in native or oxidized Lp(a)-treated EC. The effect of Lp(a)-free oxidized LDL on PAI-1 production was significantly weaker than the equimolar amount of oxidized Lp(a) but stronger than that of native LDL. Treatments with oxidized Lp(a) increased cell-associated PAI-1 to a similar extent as that in native Lp(a)-treated EC. The results of the present paper demonstrate that oxidative modification enhances Lp(a)-induced PAI-1 production in vascular endothelial cells at RNA transcription level, which suggests that oxidization potentially amplifies the anti-fibrinolytic and thrombotic effect of Lp(a).

In this study, the effects of lidocaine and hypoxia on the biosynthesis of phospholipids in the hamster heart were examined. hamster hearts were perfused with [1,3-3H]glycerol under normal and hypoxic conditions, and in the absence or presence of 0.5 mg/mL lidocaine. After perfusion, the radioactivity incorporated into the various phospholipid fractions was determined. With the exception of phosphatidylcholine, the synthesis of phospholipids was generally stimulated by lidocaine perfusion. In contrast, hypoxia caused a general decrease in phospholipid biosynthesis which was partially restored by lidocaine. ATP and CTP levels were severely reduced under hypoxic conditions, but their levels were not restored by lidocaine treatment. The activities of enzymes for phospholipid synthesis were determined under the various perfusion conditions. The activity of phosphatidic acid phosphatase was elevated by lidocaine and decreased by hypoxic treatment. The activity of CTP:phosphatidic acid cytidylyltransferase was increased under hypoxia, with or without lidocaine. Despite the reduction in phosphatidylcholine biosynthesis, no change in the activity of cytidine diphosphocholine (CDPcholine): diacylglycerol cholinephosphotransferase was detected following lidocaine or hypoxic perfusion. However, enzyme activity was inhibited by the presence of lidocaine in the assay mixture. Our results indicate that the reduction in phospholipid biosynthesis under hypoxic conditions was caused mainly by diminishing high-energy nucleotide levels. The enhancement of phospholipid biosynthesis by lidocaine appeared to be mediated in part by modulation of enzyme activities.

1. Selective blockade of platelet-activating factor (PAF) receptor subtypes by PAF receptor antagonists has been demonstrated. However, selective activation of PAF receptor subtypes by PAF receptor agonists has not been reported. 2. When structural analogues of PAF that have been shown to possess either agonist or antagonist effects were administered by a bolus injection in the rat perfused heart, they all showed agonist effects. Lower amounts produced vasodilation while higher amounts produced vasodilation followed by vasoconstriction. These coronary vascular effects were typical of that observed with PAF. Lyso-PAF did not show the same typical pattern of coronary vascular effect, confirming that the detergent effect of PAF structural analogues did not play a role in the coronary vascular effects. Other PAF antagonists, CV-6209 and WEB 2170, also did not produce the PAF-like response in the rat perfused heart. 3. The coronary vascular effects of hexanolamine-PAF (H-PAF, putative antagonist) and ethanolamine-PAF (E-PAF, agonist) were further studied. Pretreatment with FR-900452 (a PAF receptor antagonist) or MK-886 (a leukotriene synthesis inhibitor) significantly reduced the vasodilator and vasoconstrictor effects of H-PAF and E-PAF. 4. Pretreatment of rat perfused hearts with low concentrations of H-PAF and E-PAF blocked the response to PAF administration in a dose- and time-dependent manner. However, the pretreatment with either H-PAF or E-PAF did not result in a coronary vascular effect expected of a PAF receptor agonist. These results were compatible with H-PAF and E-PAF behaving as PAF receptor antagonists. 5. In summary, our results demonstrate that several PAF structural analogues possess agonist action in the rat perfused heart. Like the coronary vascular effects of PAF, the effects of H-PAF and E-PAF were blocked by a PAF antagonist (FR-900452) and a leukotriene synthesis inhibitor (MK-886). This suggests that both H-PAF and E-PAF mediate their effect through activation of PAF receptors with a subsequent release of leukotrienes that produced vasodilatation and vasoconstriction. Furthermore, pretreatment of perfused hearts with these compounds blocked the response to PAF in these hearts. Thus these compounds can also behave like a PAF receptor antagonist. This latter action may be due to a gradual receptor inactivation or desensitization by the pretreatment of H-PAF and E-PAF through a PAF receptor agonist effect rather than being a PAF receptor antagonist.

Methyl lidocaine is an experimental anti-arrhythmic drug which has been shown to enhance the biosynthesis of phosphatidyl-inositol (PI) in the hamster heart. In this study, the effect of methyl lidocaine on enzymes involved in the biosynthesis of PI in the heart was examined. When the hamster heart was perfused with labelled methyl lidocaine, the majority of the compound was not metabolized after perfusion. The direct action of methyl lidocaine on an enzyme was studied by the presence of the drug in enzyme assays, whereas its indirect action was studied by assaying the enzyme activity in the heart after methyl lidocaine perfusion. CTP:phosphatidic acid cytidylyl-transferase, a rate-limiting enzyme in PI biosynthesis, was stimulated by methyl lidocaine in a direct manner. Kinetic studies revealed that methyl lidocaine caused a change in the affinity between the enzyme and phosphatidic acid and resulted in the enhancement of the reaction. Alternatively, acyl-CoA:lysophosphatidic acid acyltransferase, another key enzyme for PI biosynthesis, was not activated by the presence of methyl lidocaine. However, the enzyme activity was stimulated in hearts perfused with methyl lidocaine. The enhancement of the acyl-transferase by methyl lidocaine perfusion was found to be mediated via the adenylate cyclase cascade with the elevation of the cyclic AMP level. The stimulation of protein kinase A activity by cyclic AMP resulted in the phosphorylation and activation of the acyltransferase. Interestingly, the activity of protein kinase C was not stimulated by methyl lidocaine perfusion. We conclude that the enhancement of PI biosynthesis by methyl lidocaine in the hamster heart resulted from the direct activation of the cytidylyltransferase, as well as the phosphorylation and subsequent activation of the acyltransferase.

Our recent study has indicated that Chinese green tea (Lung Chen), in which epigallocatechin-3-gallate (EGCG) accounts for 60% of catechins, protected cigarette smoke-induced lung injury. We now hypothesized that Lung Chen tea may also have potential effect on lung oxidative stress and proteases/anti-proteases in a smoking rat model. Sprague-Dawley rats were exposed to either sham air (SA) or 4% cigarette smoke (CS) plus 2% Lung Chen tea or water by oral gavage. Serine proteases, matrix metalloproteinases (MMPs) and their respective endogenous inhibitors were determined in bronchoalveolar lavage (BAL) and lung tissues by gelatin/casein zymography and biochemical assays. Green tea consumption significantly decreased CS-induced elevation of lung lipid peroxidation marker, malondialdehyde (MDA), and CS-induced up-regulation of neutrophil elastase (NE) concentration and activity along with that of α(1)-antitrypsin (α(1)-AT) and secretory leukoproteinase inhibitor (SLPI) in BAL and lung. In parallel, significant elevation of MMP-12 activity was found in BAL and lung of the CS-exposed group, which returned to the levels of SA-exposed group after green tea consumption but not CS-induced reduction of tissue inhibitor of metalloproteinase (TIMP)-1 activity, which was not reversed by green tea consumption. Taken together, our data supported the presence of local oxidative stress and protease/anti-protease imbalance in the airways after CS exposure, which might be alleviated by green tea consumption through its biological antioxidant activity.

Lidocaine is used clinically as an antiarrhythmic agent, but its effect on cardiac phospholipid metabolism has not been defined. In this study, hamster hearts were perfused with [1,3-3H]glycerol in the presence of 0.5 mg/mL lidocaine. The incorporation of radioactivities into lysophosphatidic acid, phosphatidic acid, phosphatidylethanolamine, cytidine diphosphate diacylglycerol, phosphatidylinositol, phosphatidylserine, diacylglycerol and triacylglycerol were enhanced by lidocaine treatment, whereas the labelling of phosphatidylcholine was reduced. Analyses of enzyme activities in the heart after perfusion with lidocaine revealed that the activities of phosphatidate phosphatase and acyl-coenzyme A (CoA):1,2-diacylglycerol acyltransferase were enhanced. The presence of lidocaine in the assay did not directly stimulate these enzymes. However, the activity of acyl-CoA:glycerol-3- phosphate acyltransferase was stimulated by lidocaine whereas the activity of cytidine diphosphocholine:1,2-diacylglycerol cholinephosphotransferase was inhibited by lidocaine. We conclude that lidocaine affects the regulation of phospholipid biosynthesis in the heart by both direct and indirect modulation of phospholipid biosynthetic enzymes.

Objective: The aim was to examine the effect of selenium supplement on endothelium dependent relaxation in rat aortic rings. Methods: Rats were supplemented with selenium for 3 d (intraperitoneal injection of 4.33 μmol sodium selenite·kg−1 body weight·d−1). Saline injections served as controls. Rat aortic ring was precontracted with phenylephrine and endothelium dependent relaxation was produced by the addition of acetylcholine. Results: Acetycholine-induced endothelium dependent relaxation was enhanced in aortic rings from rats after receiving selenium supplement as compared to control rats. For comparison, endothelium independent vasodilators (sodium nitroprusside and cromakalim) were investigated. Selenium supplement did not affect the relaxation produced by these vasodilators. N-nitro-L-arginine methyl ester (L-NAME) abolished acetylcholine induced relaxation in aortic rings from animals with selenium supplement while indomethacin had no effect on the relaxation. Direct addition of selenium or glutathione peroxidase and glutathione into the organ bath had no effect on acetylcholine induced endothelium dependent relaxation. Conclusions: The enhanced relaxation after selenium supplement was due to an increase in the release of nitric oxide since L-NAME was effective in blocking the relaxation. The lack of an effect by indomethacin indicated little role for cyclo- oxygenase products in this system. Our results suggest that the effect of selenium supplement on endothelium dependent relaxation is mediated by cellular changes that cannot be mimicked by the direct addition of selenium or the selenium dependent enzyme glutathione peroxidase. Cardiovascular Research 1994; 28 :345-348

Previous studies have shown the pathogenic effects of grains cultivated in the endemic areas of Keshan disease and selenium is effective in the prevention of this disease. In this study, liver damages induced by feeding grains from an endemic area (endemic diet), and the effects of selenium and α-tocopherol supplement were examined. After 3 months on the endemic diet, the amounts of serum enzymes were significantly increased when compared to controls (animals receiving diet from a non-endemic area). Liver enzymes (alkaline phosphatase and choline esterase) were also found to be altered in the serum, further suggesting liver damages in animals on an endemic diet. Supplement of the endemic diet with selenium or α-tocopherol reversed the changes in serum enzymes. Increase in lipid peroxidation in the liver of animals on the endemic diet was observed when compared to that in control animals. Selenium and α-tocopherol supplements prevented the increase in lipid peroxidation in the liver by the endemic diet. Semi-quantitative histochemical analysis of glutamate dehydrogenase and succinate dehydrogenase in liver tissue showed that the livers of animals on an endemic diet were more sensitive to ischemic damagesin vitro. Supplementation of the endemic diet with either selenium or α-tocopherol reduced the sensitivity to ischemic damages. The results suggest that increased lipid peroxidation in the liver of rats on an endemic diet may be responsible for liver damages and elevation of serum enzymes. Restoration of glutathione peroxidase activity by selenium supplement or an increase in the content of α-tocopherol in the liver can prevent lipid peroxidation in animals on an endemic diet and thus provide the protective effects against liver damages.

The present study investigated the mechanism underlying the transient potentiation of vasoconstriction by hypoxia in isolated porcine coronary arteries. Isometric tension was measured in rings with or without endothelium. Hypoxia (Po2 ω-nitro-l-arginine methyl ester (l-NAME)] or soluble guanylyl cyclase (ODQ and NS2028). The addition of DETA NONOate (nitric oxide donor) in the presence of l-NAME restored the hypoxic augmentation, suggesting the involvement of the nitric oxide pathway. However, the same was not observed after incubation with 8-bromo-cyclic GMP, atrial natriuretic peptide, or isoproterenol. Assay of the cyclic GMP content showed no change upon exposure to hypoxia in preparations with and without endothelium. Incubation with protein kinase G and protein kinase A inhibitors did not inhibit the hypoxic augmentation. Thus the hypoxic augmentation is dependent on nitric oxide and soluble guanylyl cyclase but independent of cyclic GMP. The hypoxic augmentation persisted in calcium-free buffer and in the presence of nifedipine, ruling out a role for extracellular calcium influx. Hypoxia did not alter the intracellular calcium concentration, as measured by confocal fluorescence microscopy. This observation and the findings that hypoxic augmentation is enhanced by thapsigargin (sarco/endoplasmic reticulum calcium ATPase inhibitor) and inhibited by HA1077 or Y27632 (Rho kinase inhibitors) demonstrate the involvement of calcium sensitization in the phenomenon.

The aim of the present study was to investigate the signaling mechanisms underlying the non-genomic effects of estrogen in rat superior mesenteric arteries. Isometric tension was recorded in rings with or without endothelium. Changes in cyclic nucleotide levels and protein kinase (PK) activities were measured. Localization of estrogen receptors (ER) and caveolin-1 were visualized by confocal microscopy. 17β-Estradiol elicited a concentration-dependent relaxation. The relaxation was reduced by SQ 22536 (adenylyl cyclase inhibitor) and KT 5823 (PKG inhibitor) while ODQ (guanylyl cyclase inhibitor) and KT 5720 (PKA inhibitor) had no effect. At the physiological concentration of 1 nM, 17β-estradiol had no significant effect on relaxation but enhanced the relaxation to sodium nitroprusside. The enhancement of relaxation by 17β-estradiol was blocked by SQ 22536 and KT 5823. Although 1 nM 17β-estradiol or 10 nM sodium nitroprusside given alone had minimal effects on PKG activity, in their combined presence, a significant increase in PKG activity was observed. Confocal microscopy demonstrated that ERα and ERβ colocalized with caveolin-1 and PKG in vascular smooth muscle cells. The present findings suggest that 17β-estradiol enhances relaxation of vascular smooth muscle of the rat superior mesenteric artery by activating adenylyl cyclase, leading to an increase in cAMP which cross activates PKG in the caveolae. No detectable increase in total cAMP level was detected as these changes occurred in the caveolae. These results are consistent with the notion that 17β-estradiol mediates its effect in the distinct microdomains of the caveolae of the plasma membrane with colocalization of adenylyl cyclase and PKG.

The effect of selenium supplement on glycerolipid biosynthesis in the isolated rat heart was investigated. Selenium was administered to the rat by intraperitoneal injection of 4.33 mumol/kg per day for 3 consecutive days. Animals administered with an equal volume of saline were used as controls. Hearts from both animal groups were perfused in Krebs-Henseleit buffer containing labelled glycerol. Subsequent to perfusion, the radioactivity associated with each glycerolipid group was determined. Selenium supplement caused elevations in the labelling of phosphatidic acid and phosphatidylcholine but not in other phospholipids, diacylglycerol or triacylglycerol. The mechanisms for the enhancement of labelling into phosphatidic acid and phosphatidylcholine were examined. The activity of the enzymes responsible for the synthesis of phosphatidic acid in the rat heart was not changed by selenium supplement. However, a 51% increase in the acyl-CoA level was detected which might account for the elevated labelling of phosphatidic acid in the selenium supplemented animal. The 2-fold increase in the activity of CDPcholine:diacylglycerol cholinephosphotransferase might also account for the increase in the labelling of phosphatidylcholine in the heart of the selenium-supplemented rat. It is clear from this study that selenium plays a regulatory role in the control of cellular lipid metabolism.

The Experimental Biology 2010, Anaheim, CA., 24-28 April 2010. In The FASEB Journal, 2010, v. 24 Meeting abstract suppl., abstract no. 960.2

Methyl-lidocaine is an amphiphilic agent which has been used as an experimental anti-arrhythmic drug. When hamster hearts were perfused with labelled glycerol, the presence of methyl-lidocaine in the perfusate was found to enhance the labelling in phosphatidylserine, phosphatidylinositol, diacylglycerol and triacylglycerol. However, the labelling of phosphatidylcholine and phosphatidylethanolamine was not significantly changed by methyl-lidocaine treatment. Assays in vitro for the enzymes involved in the synthesis of neutral lipids and acidic phospholipids revealed that phosphatidate phosphatase and CTP: phosphatidate cytidylyltransferase activities were stimulated by methyl-lidocaine. The intracellular pool sizes of diacylglycerol and CDP-diacylglycerol were also elevated. We postulate that the enhanced syntheses of the neutral lipids and acidic phospholipids in the methyl-lidocaine-perfused heart were mediated via the direct activation of the key enzymes in the biosynthesis of these lipids de novo.

Objective To explore the effects of different estrogens on vascular function, we compared the vasorelaxant effects of 17beta-estradiol, 17alpha-estradiol, estrone, and the synthetic estrogen diethylstilbestrol (DES) on porcine coronary arterial segments. Design Porcine coronary arterial rings were contracted with the stable thromboxane A2 analogue U46619 (3 x 10(-8) M), and direct relaxation was examined by the addition of increasing concentrations of 17beta-estradiol, 17alpha-estradiol, estrone, or DES (10(-9) to 10(-4) M). Modulation of agonist-induced contraction and relaxation was studied in coronary arterial rings incubated for 20 minutes with DES or estrone (10(-10)-10(-6) M) with 17beta-estradiol (10(-9) M) as comparison. Results Direct relaxation of arterial rings potentiated by these estrogens was recorded with a rank order potency of DES > 17beta-estradiol > estrone > 17alpha-estradiol. 17beta-Estradiol potentiated relaxation responses to sodium nitroprusside and levcromakalim but not bradykinin or A23187 while reducing contractions to 5-hydroxytryptamine and U46619. DES and estrone, both at 10(-6) M, mimicked the 17beta-estradiol-potentiated sodium nitroprusside and levcromakalim relaxation responses. Additionally, the inhibitory effects of 17beta-estradiol (10(-9) M) on 5-hydroxytryptamine- and U46619-induced contractions were partially reproducible by DES (10(-6) M) and estrone (10(-6) M). Conclusions Although DES is the most potent among the tested estrogenic compounds in eliciting relaxation, 17beta-estradiol is more effective than estrone and DES at enhancing endothelium-independent relaxation and reducing vascular contraction in porcine coronary arteries.

Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse in vivo and in vitro coronary vascular effects. In the present study, the coronary vascular responses to bolus injections of PAF were compared in rat hearts perfused under constant flow and under constant pressure. Low levels of PAF (1 pmol) produced vasodilatation only, while higher PAF concentrations (100 pmol) produced initial vasodilatation which was followed by a vasoconstriction under both experimental conditions. To determine species differences in PAF action, the effect of PAF was tested on perfused guinea pig hearts. Unlike in perfused rat hearts, only a dose-dependent vasoconstrictor response was observed in perfused guinea pig hearts following a bolus injection of 1 fmol to 10 pmol of PAF. The results from repeated injections of PAF indicated that depletion of vasoactive mediators induced by PAF or receptor desensitization may explain a failure of a second injection of PAF to initiate a vasoconstrictor response. After PAF injection, the coronary vascular response to leukotriene was not altered, indicating that the reduced vasoconstrictor effect of a second injection of PAF cannot be due to a reduced ability of the smooth muscle to constrict. The study demonstrates that similar coronary vascular responses to PAF are observed in perfused rat hearts under either constant flow rate or constant pressure and that some of the variable coronary vascular responses reported may be due to the difference between animal species.

Lysophosphatidylcholine (lysoPC) is an arrhythmogenic phospholipid metabolite which accumulates in the ischemic myocardium. Reduced catabolism of lysoPC has been proposed to be one of the biochemical mechanisms responsible for the increase in lysoPC content. In this investigation we compared the microsomal catabolism of exogenous labeled lysoPC in isolated perfused rat and guinea pig hearts. Analysis of the amount of radioactivity in microsomal phosphatidylcholine (PC) and free fatty acid (FFA) was used as an index of the participation in lysoPC clearance by acylation catalyzed by acyl-CoA:lysoPC acyltransferase and deacylation catalyzed by lysophospholipase, respectively. There was no significant difference in the incorporation of radioactivity into rat and guinea pig heart microsomes; however, the patterns of radioactivity in lysoPC metabolites were notably different. Equal participation by deacylation and reacylation was observed in rat microsomes, whereas deacylation was clearly the preferred route for lysoPC clearance in guinea pig microsomes. Modulation of enzyme activity by treatment of the isolated heart with pHMB, a sulfhydryl agent, was used to probe the relationship among acylation, deacylation and the extent of lysoPC clearance. In guinea pig microsomes impairment of lysoPC acylation was not associated with any change in the amount of radioactivity in lysoPC because of a compensatory increase in deacylation. In contrast, impaired deacylation in rat microsomes led to significant elevations in the amount of radioactivity in lysoPC. We conclude, therefore, that in intact perfused rat and guinea pig hearts the relative participation of acylation and deacylation in lysoPC clearance differs. Moreover, we propose that the level of deacylation by lysophospholipase is an important factor in the extent of clearance of lysoPC.

1. In a previous study it was demonstrated that bolus injections of platelet-activating factor (PAF) in the rat perfused heart resulted in coronary vasodilatation, vasoconstriction or the combination of both, depending on the amount of PAF that was injected. In the present study, the mechanisms of these coronary vascular effects of PAF in the rat perfused heart were investigated. 2. Pretreatment of the rat perfused heart with the PAF antagonists FR-900452 or BN-52021 did not affect the vasodilator effect of PAF but eliminated the vasoconstrictor effect of PAF. FR-900452 had no effect on the vasoconstrictor response to leukotriene C4 (LTC4) or LTD4. 3. The cyclo-oxygenase inhibitor, indomethacin, did not modify the coronary vascular effects of PAF. However L-649,923 (a leukotriene antagonist) and MK-886 (a leukotriene synthesis inhibitor) eliminated both the vasodilator and vasoconstrictor effects of PAF. 4. When leukotrienes were administered by bolus injection in the rat perfused heart, LTB4 produced vasodilatation while LTC4 and LTD4 produced vasoconstriction. L-649,923 blocked both the vasodilator and vasoconstrictor effects of the leukotrienes tested. 5. The results suggest that lipoxygenase products are responsible for both the vasodilator and vasoconstrictor actions of PAF in the coronary vasculature of the rat perfused heart while the cyclo-oxygenase products do not play a significant role. The ineffectiveness of PAF antagonists in blocking the vasodilatation produced by PAF is compatible with the concept that there may be multiple PAF receptors.

"Danshen" has been used for the treatment of various cardiovascular diseases in the People's Republic of China for many years. Two different forms of "Danshen" exist, with the roots of Salvia miltiorrhiza being the traditional form and the roots of Salvia przewalskii being a surrogate used in the western areas of mainland China. The most abundant lipophilic diterpene quinones present in S. miltiorrhiza and S. przewalskii roots, tanshinone IIA (1) and cryptotanshinone (2), inhibited contraction of the isolated porcine coronary artery to the thromboxane A(2) analogue, U46619. Przewaquinone A (3), a lipophilic diterpene quinone present only in S. przewalskii, induced a similar but greater inhibitory action on vascular contraction than 1 and 2. This effect of 3 was endothelium-independent and reversible. The present results suggest that 3 is more potent than 1 and 2 and may contribute to a great extent to the ability of S. przewalskii roots to inhibit vascular contractions.