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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.

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.

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.

The present experiments investigated whether endothelium-derived mediators modulate the effect of natriuretic peptides in porcine coronary arteries. Rings with and without endothelium were suspended in organ chambers for isometric tension recording. Concentration-relaxation curves to C-type natriuretic peptide (CNP) and atrial natriuretic peptide (ANP) were obtained during contractions to endothelin-1. Removal of the endothelium potentiated relaxations to both CNP and ANP. N(omega)-nitro-L-arginine methyl ester potentiated relaxations to natriuretic peptides only in arteries with endothelium. Sodium nitroprusside (SNP) inhibited the response to the natriuretic peptides only in the absence of the endothelium. In rings with endothelium, 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (NS2028) potentiated CNP-mediated relaxations. Iberiotoxin (IBTX) reduced the response only in rings without endothelium. Glybenclamide inhibited the relaxations in both the presence and absence of endothelium. CNP-induced relaxations were reduced by 8-bromoguanosine 3',5'-cGMP (8-bromo-cGMP) to the same extent in rings with and without endothelium. There was no significant difference between the increased cGMP content caused by CNP in porcine coronary arteries with or without endothelium. In patch-clamp studies in porcine coronary arterial smooth muscle cells, the natriuretic peptide-mediated enhancement of the IBTX-sensitive big conductance calcium-activated potassium channel (BK(Ca)) amplitude was reversed by SNP and 8-bromo-cGMP. These findings demonstrate that, in the porcine coronary artery, the opening of BK(Ca) and ATP-dependent potassium channels of the vascular smooth muscle contributes to CNP-mediated relaxations. Endothelium-derived and exogenous NO inhibit the direct relaxing effect of natriuretic peptides by desensitizing the response of the BK(Ca)s of the vascular smooth muscle to the generation of cGMP.

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

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

The present experiments were designed to determine whether or not endothelium-dependent contractions can be evoked in the aorta of the mouse, and if so, whether or not deleting the COX1 gene affects the response. Sex differences in the response were also examined. Rings of murine aorta were suspended in a Halpern-Mulvany myograph for recording of isometric force. In the aorta of the male wild type C57BL/b6 mice (36-40 weeks old), both acetylcholine and the calcium ionophore caused endothelium-dependent increases in force in the presence of L-NAME, and these were inhibited by valeryl salicylate (a selective COX1 inhibitor) and S18886 (a selective antagonist of TP receptors). Such endothelium-dependent contraction was absent in the aorta of COX1 knockout mice and present in that of COX2 knockout mice. Similar results were obtained in aortas of female wild-type, COX2 and COX1 knockout mice. These experiments reveal the existence of EDCF-mediated contractions in arteries of the mouse. These contractions, as in the aorta of the spontaneously hypertensive rat, are caused by endogenous agonists(s) of TP receptors produced by cyclooxygenase 1, because they are observed in the aortas of COX2 knockout mice but not in aortas of COX1 knockout mice. The present study provides direct evidence that COX1 is indeed the isoform of cyclooxygenase responsible for the production of EDCF.

The aim of the present study was to investigate the gender differences in the acute effects of 17β-estradiol on the rat superior mesenteric artery. Isometric tension was measured in rings of mesenteric arteries from both male and female Sprague–Dawley rats. Relaxation to acetylcholine was not significantly different between arteries (with endothelium) from male and female rats in the absence or presence of 17β-estradiol. After blockade of endothelium-dependent hyperpolarizations with apamin (0.3 μM) plus charybdotoxin (0.1 μM), acute exposure to 17β-estradiol (1 nM) for 30 min resulted in enhancement of relaxation to acetylcholine in arteries from male but not female rats. After acute exposure to 17β-estradiol, mesenteric arteries from male rats were more sensitive to sodium nitroprusside than arteries from female rats. Contractions of mesenteric arteries to phenylephrine and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619) were greater in arteries from male rats than female rats. This difference was not detected after acute exposure to 17β-estradiol. In preparations without endothelium, the enhancement of relaxation and reduction in contraction in arteries from male rats were preserved. These results suggest that there exists a gender difference in the response to the acute nongenomic modulatory effect of 17β-estradiol in rat mesenteric arteries. Arteries from male rats seem to be more sensitive to the modulatory effects of 17β-estradiol than arteries from female rats. The effect appears to be mainly at the level of the vascular smooth muscles. British Journal of Pharmacology (2005) 146, 1148–1155. doi:10.1038/sj.bjp.0706422

The etiology of the atherosclerosis that occurs in diabetes mellitus is unclear. Adenosine has been shown to inhibit growth of rat aortic smooth muscle cells. Nucleoside transporters play an integral role in adenosine function by regulating adenosine levels in the vicinity of adenosine receptors. Therefore, we studied the effect of 25 mM d-glucose, which mimics hyperglycemia of diabetes, on adenosine transport in cultured human aortic smooth muscle cells (HASMCs). Although RT-PCR demonstrated the presence of equilibrative nucleoside transporter-1 (ENT-1) and ENT-2 mRNA, functional studies revealed that adenosine transport in HASMCs was predominantly mediated by ENT-1 and inhibited by nitrobenzylmercaptopurine riboside (NBMPR, IC50 = 0.69 ± 0.05 nM). Adenosine transport in HASMCs was increased by >30% after treatment for 48 h with 25 mM d-glucose, but not with equimolar d-mannitol and l-glucose. Kinetic studies showed that d-glucose increased Vmax of adenosine transport without affecting Km. Similarly, d-glucose increased Bmax of high-affinity [3H]NBMPR binding, while the dissociation constant ( Kd) was not changed. Consistent with these observations, 25 mM d-glucose increased mRNA and protein expression of ENT-1. Treatment of serum-starved cells with the selective inhibitors of MAPK/ERK, PD-98059 (40 μM) and U-0126 (10 μM), abolished the effect of d-glucose on ENT-1. We conclude that d-glucose upregulates the protein and message expression and functional activity of ENT-1 in HASMCs, possibly via MAPK/ERK-dependent pathways. Pathologically, the increase in ENT-1 activity in diabetes may affect the availability of adenosine in the vicinity of adenosine receptors and, thus, alter vascular functions in diabetes.

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.

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.

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

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.

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.

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

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.

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.

The involvement of ecto-5′-nucleotidase (E-5′Nu) in the elevation of extracellular adenosine during inflammation is unclear. In the present study, the effect of lipopolysaccharide (LPS), an inflammation inducer, was investigated on E-5′Nu in human umbilical vein endothelial cells (HUVECs). E-5′Nu activity was enhanced after a 24 h exposure to LPS. This effect was dose dependent, with an EC50 of 1.66 ng/ml. At 10 μM, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 abolished the LPS-induced E-5′Nu activity. However, at 10 μM, the NF-κB inhibitor ammonium pyrrolidine dithiocarbamate had no effect. LPS upregulated the protein expression but not the messenger RNA expression of E-5′Nu. The inhibition of E-5′Nu by 100 μM α,β-methylene adenosine-5′-diphosphate increased the LPS-induced inflammation, suggesting that E-5′Nu plays a significant role in reducing inflammation, probably through the generation of adenosine. In conclusion, the experiments indicate that LPS upregulates E-5′Nu activity in HUVECs through a PI3K-dependent increase in the abundance of E-5′Nu on cell membranes. Since adenosine is an anti-inflammatory molecule, E-5′Nu upregulation may be crucial in protecting endothelial cells against inflammatory damage.

The available evidence suggests that vitamin D has cardiovascular effects besides regulating calcium homeostasis. To examine the effect of 1,25-dihydroxyvitamin D3, the major metabolite of vitamin D, on endothelium-dependent contractions, aortic rings of spontaneously hypertensive rats (SHR) were suspended in organ chambers for isometric force measurements. Rings were incubated with Nω-nitro-l-arginine methyl ester (l-NAME) and then exposed to increasing concentrations of acetylcholine, ATP, or the calcium ionophore to trigger contractions. This was done in the absence or presence of 1,25-dihydroxyvitamin D3. The release of prostacyclin after acetylcholine or A-23187 stimulation was also measured. The cytosolic-free calcium concentration was measured by confocal microscopy after incubation with the fluorescent dyes fluo-4 and fura red. The presence of vitamin D receptors was confirmed using immunohistochemistry. Acetylcholine- and ATP-induced endothelium-dependent contractions were significantly reduced compared with those obtained in the absence of the drug. This effect was not present if A-23187 was used as an agonist. The acetylcholine- but not the A-23187-induced release of prostacyclin was reduced by the acute administration of 1,25-dihydroxyvitamin D3. Exposure to 1,25-dihydroxyvitamin D3 reduced the increase in cytosolic-free calcium concentration caused by acetylcholine but not by A-23187 in cells. Vitamin D receptors were densely distributed in the endothelium. Inecalcitol (19-nor-14-epi-23-yne-1,25-dihydroxyvitamin D3), a synthetic analog of vitamin D, caused a comparable depression of endothelium-dependent contractions as 1,25-dihydroxyvitamin D3. These results demonstrate that vitamin D3 modulates vascular tone by reducing calcium influx into the endothelial cells and hence decreasing the production of endothelium-derived contracting factors.

Study objective – The aim was to evaluate the arrhythmogenic effect of a free radical generating system, FeCl3/ADP, using two different approaches. Design – Ventricular arrhythmias were studied in isolated rat hearts subjected to regional ischaemia and reperfusion without or with simultaneous treatment with nicergoline (0.4 mg·litre−1). In the second part of this study the electrophysiological effects of FeC13/ADP (0.1/1.0 μM) were investigated in normal Purkinje fibres and in Purkinje fibres from dog surviving infarction, by using conventional microelectrode method. Experimental materials – Hearts were obtained from male Sprague-Dawley rats, weight 250-300 g. Purkinje fibres were dissected from hearts of mongrel dogs of either sex (10-15 kg) with or without prior myocardial infarction. Measurements and results – FeC13/ADP (0.1/1.0 μM and 1.0/1.0 μM respectively) weakly changed the incidence of reperfusion induced arrhythmias. In nicergoline pretreated hearts, in which the incidence of reperfusion arrhythmias was reduced, FeC13/ADP (0.1/1.0 μM and 1.0/1.0 μM) did not change the incidence and the duration of reperfusion arrhythmias. In normal Purkinje fibres, FeC13/ADP (0.1/1.0 μM) induced a decrease in action potential duration without any pronounced effect on Vmax, diastolic potential, and activation potential. In Purkinje fibres from post infarct myocardium, FeCl3/ADP decreased action potential duration, diastolic potential, and activation potential. Conclusions – Free radical generation did not antagonise the antiarrhythmic activity of α adrenergic blockade. Free radical generation induced slow and minor changes in electrophysiological activity of Purkinje fibres both from normal and ischaemic hearts. Our data suggest that free radical generation may not be the only mechanism involved in the genesis of reperfusion arrhythmias.

Study objective – The aim of the study was to investigate the electrophysiological effects of acetylcholine in Purkinje fibres surviving infarction. Design – Infarction in canine hearts was produced by coronary occlusion. Ischaemic tissue was removed and surviving Purkinje fibres were compared for effects of increasing concentrations of acetylcholine (1-100 μM) with control Purkinje fibres from normal hearts and normal Purkinje fibres treated with barium. Subjects – Experimental animals were mongrel dogs of either sex, weight 10-15 kg, n = 14 (control), 9 (infarction group), 11 (Ba treated group). Measurements and main results – Acetylcholine caused concentration dependent shortening of action potential duration in normal Purkinje fibres and in fibres surviving infarction, but had no effect when potassium conductance was decreased with Ba. Acetylcholine induced a small hyperpolarisation in normal Purkinje fibres and in fibres surviving infarction perfused with a 4 mM potassium solution without affecting action potential amplitude or dV/ dtmax. Reduction in K concentration to 1 and 2 mM caused a greater depolarisation of diastolic potential in normal fibres than in fibres surviving infarction or Ba treated fibres. Acetylcholine produced hyperpolarisation under these conditions in normal fibres only. Acetylcholine decreased automaticity in normal fibres only at high concentration (100 μM). A lesser effect was seen in Ba treated fibres and in fibres surviving infarction. Conclusions – Since Ba treated Purkinje fibres had similar action potential characteristics to those seen in Purkinje fibres surviving infarction and responded in the same way to the modifications of K concentration, a decrease in K conductance might be the underlying mechanism for some of the electrophysiological changes in fibres surviving infarction. The results also suggest that after 24 h ischaemia, surviving Purkinje fibres have a different sensitivity to acetylcholine from normal fibres.

Aims The present study examined the hypothesis that prostaglandin E2 (PGE2) through activation of prostaglandin E (EP) receptor contributes to endothelium-dependent contractions. Methods and results Western blotting revealed that the protein expression of EP1 receptor was significantly down-regulated in the aorta of the spontaneously hypertensive rat (SHR), but there was no significant difference in the expression of EP2, EP4, and total EP3 receptors between preparations of Wistar Kyoto rats (WKY) and SHR. Isometric tension studies showed that low concentrations of PGE2 caused endothelium-dependent relaxations in WKY but not in aortas of the SHR. High concentrations of PGE2 evoked contractions predominately through the activation of thromboxane-prostanoid (TP) receptors in the WKY, but involves the dual activation EP and TP receptors in the SHR. SQ29,548, BAYu3405 and Terutroban (TP receptor antagonists), and AH6809 (non-selective EP receptor antagonist) abolished, while SC19220 (preferential EP1 receptor antagonist) did not inhibit endothelium-dependent contractions. Both SC19220 and AH6809 significantly inhibited contractions to U46619 (TP receptor agonist). Conclusion The present study demonstrates that the contraction caused by PGE2 in the SHR aorta is dependent on the activation of EP1 and TP receptors, but that endothelium-dependent contractions do not require the former. Thus, PGE2 is unlikely to be an endothelium-derived contracting factor in this artery. The ability of AH6809 to inhibit endothelium-dependent contractions can be attributed to its partial antagonism at TP receptors. Nevertheless, the impairment of PGE2-mediated relaxation may contribute to endothelial dysfunction in the aorta of the SHR.

Dihydropyridine-type calcium channel antagonists, in addition to having a vasodilatory effect, are known to inhibit cellular uptake of nucleosides such as adenosine. However, the nucleoside transporter subtypes involved and the mechanism by which this occurs are not known. Therefore, we have studied the inhibitory effects of dihydropyridines on both human equilibrative nucleoside transporters, hENT-1 and hENT-2, which are the major transporters mediating nucleoside transport in most tissues. Among the dihydropyridines tested, nimodipine proved to be the most potent inhibitor of hENT-1, with an IC(50) value of 60+/-31 muM, whereas nifedipine, nicardipine, nitrendipine, and felodipine exhibited 100-fold less effective inhibitory activity. Nifedipine, nitrendipine, and nimodipine inhibited hENT-2 with IC(50) values in the micromolar range; however, nicardipine and felodipine had no significant effect on hENT-2. Removal of the 4-aryl ring or changing the nitro group at the 4-aryl ring proved not to be detrimental to the inhibitory effects of dihydropyridines on hENT-1, but resulted in a drastic decrease in their inhibitory effects on hENT-2. Kinetic studies revealed that nimodipine and nifedipine reduced V(max) of [(3)H]uridine transport without affecting K(m). The inhibitory effects of nimodipine and nifedipine could be washed out. In addition, nimodipine and nifedipine inhibited the rate of NBTGR-induced dissociation of [(3)H]NBMPR from hENT-1 cell membrane. We conclude that dihydropyridines are non-competitive inhibitors of hENT-1 and hENT-2, probably working through reversible interactions with the allosteric sites. The inhibitory potencies of dihydropyridines may be associated with the structure of the 4-aryl ring, as well as the ester groups at the C-3 and C-5 positions.

Puerarin, an isoflavonoid derived from the Chinese medicinal herb Radix puerariae, has been suggested to be useful in the management of various cardiovascular disorders. The present study examined the effect of acute exposure (30 min) to puerarin on vascular relaxation. Rings from porcine coronary artery of either sex were used. The highest concentration of puerarin (100 microM) produced a small but statistically significant relaxation of U46619-contracted rings. Vascular relaxations were also studied in the presence of lower concentrations of puerarin (0.1, 1 and 10 microM) which had no direct relaxation effect. Puerarin enhanced vasorelaxation to endothelium-independent relaxing agents, sodium nitroprusside and cromakalim. However, puerarin had no effect on vasorelaxation induced by endothelium-dependent relaxing agents, bradykinin and calcium ionophore A23187. The potentiating action of puerarin (10 microM) on sodium nitroprusside-mediated relaxation was not affected by the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 microM), or by the disruption of the endothelium with Triton X-100. The effect of puerarin was reversible following a washout period. The potentiating effects were comparable with the 3'-5'-cyclic adenosine monophosphate (cyclic AMP) analogues, 8-bromoadenosine-3'-5'-cyclic monophosphate (8-Br-cyclic AMP; 10 muM) and Sp-isomer [S nomenclature refers to phosphorus] of adenosine-3', 5'-cyclic monophosphorothioate (Sp-cyclic AMPS; 3 microM), but not the 3'-5'-cyclic guanosine monophosphate (cyclic GMP) analogue, 8-bromoguanosine-3'-5'-cyclic monophosphate (8-Br-cyclic GMP; 3 microM). The cyclic AMP antagonist, Rp-isomer [R nomenclature refers to phosphorus] of 8-bromoadenosine-3', 5'-cyclic monophosphorothioate (Rp-8-Br-cyclic AMPS; 10 microM), but not cyclic GMP antagonist, Rp-isomer of 8-bromoguanosine-3', 5'-cyclic monophosphorothioate (Rp-8-Br-cyclic GMPS; 10 microM), reversed the effects of puerarin (10 microM) on the enhancement of vasorelaxation to sodium nitroprusside. Our results demonstrated that puerarin enhanced sodium nitroprusside-induced relaxation, possibly via the cyclic AMP-dependent pathway.

Individual vascular beds exhibit differences in vascular reactivity. The present study investigates the effects of streptozotocin-induced type I diabetes on endothelium-dependent responses of rat carotid, femoral, and mesenteric arteries. Rings with and without endothelium, suspended in organ chambers for isometric tension recording, were contracted with phenylephrine and exposed to increasing concentrations of acetylcholine. In carotid and femoral arteries, acetylcholine produced concentration- and endothelium-dependent relaxations that were abolished by Nomega-nitro-L-arginine methyl ester (L-NAME; specific nitric-oxide synthase inhibitor) and were impaired slightly in preparations from streptozotocin-treated rats (STZ-rats). This impairment could be prevented by L-arginine. In femoral arteries incubated with L-NAME, acetylcholine caused endothelium-dependent contractions that were abolished by 3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronapht]-1-yl) propionic acid (S18886) (antagonist of thromboxane A2/prostaglandins H2-receptors) and reversed to relaxation by indomethacin (inhibitor of cyclooxygenase). The latter relaxation was inhibited by charybdotoxin plus apamin, suggesting a role of endothelium-dependent hyperpolarizing factor (EDHF). This EDHF-mediated component was augmented slightly in arteries from STZ-rats. In mesenteric arteries, relaxations to acetylcholine were only partially inhibited by L-NAME, and the L-NAME-resistant component was abolished by charybdotoxin plus apamin. In the mesenteric arteries from STZ-rats, L-NAME-sensitive relaxations to acetylcholine were reduced and the EDHF-component was augmented. These findings demonstrate a marked heterogeneity in endothelium-dependent responses in rat arteries and their differential adaptation in the course of type I diabetes. In particular, the EDHF-mediated component not only compensates for the reduced bioavailability of nitric oxide in the femoral and mesenteric artery but also counteracts the augmented endothelium-dependent contractions in the former.

Adenosine plays a significant role in various physiological processes including cardioprotection. Nucleoside transporters modulate adenosine levels in the vicinity of adenosine receptors, which in turn modulate adenosine functional efficacy. In the current study, adenosine transport in the rat heart myoblast cell line H9c2 was characterized. Kinetic analysis of adenosine transport in H9c2 cells revealed a K m of 8.9±0.001 μM and a V max of 32.1±0.65 pmol/mg protein/min. Adenosine transport in H9c2 cells was Na + -independent. About 6% of the total adenosine uptake was sensitive to nitrobenzylmercaptopurine riboside (NBMPR); however, 94% was insensitive, suggesting that adenosine uptake by H9c2 cells was predominantly mediated by the equilibrative nucleoside transporter (ENT)-2 and only mildly by ENT-1. Results of RT–PCR demonstrated the presence of mRNA for ENT-1, ENT-2 and ENT-3. Upon culture in a cell differentiation medium containing fetal bovine serum (1%) and retinoic acid (10 nM), both the activity and mRNA expression of ENT-1 increased 3-fold, however, ENT-2 was unaffected. Pharmacological studies revealed that ENT-1 activity was stimulated by PKA and PKC-δ/e, however, ENT-2 activity was unaffected. Taken together, the exceptionally high expression level of ENT-2 in H9c2 cells raises questions regarding the use of H9c2 cells as a model for physiological adenosine activity in the heart. Furthermore, this study may form the basis for further investigation into the effect of cell differentiation and protein kinases on the regulation of nucleoside transporters.

Preliminary studies suggested that previous exposure to acetylcholine (ACh) exerts a delayed inhibition of subsequent contractions mediated by endothelium-derived contracting factor (EDCF). To confirm this long-term inhibitory effect of ACh and to determine whether nitric oxide (NO) mediates the phenomenon, we suspended rings of spontaneously hypertensive rat (SHR) aortas in organ chambers for the recording of isometric force. The rings were incubated in the absence or presence of Nω-nitro-l-arginine methyl ester (l-NAME; inhibitor of NO synthases) or 1 H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ; inhibitor of soluble guanylyl cyclase) before exposure to increasing concentrations of ACh or sodium nitroprusside (SNP) during contractions to phenylephrine. Thereafter, EDCF-mediated contractions to ACh or the calcium ionophore A-23187 were elicited. If the rings were preexposed to ACh or SNP, the subsequent ACh-induced EDCF-mediated contractions were reduced compared with those obtained in rings of the same arteries not previously exposed to either agent. ODQ did not affect the inhibition caused by preexposure to ACh but significantly reduced that caused by preexposure to SNP. Previous exposure to SNP reduced, whereas previous exposure to ACh did not affect, endothelium-dependent contractions to A-23187. Previous exposure to either ACh or SNP did not affect the contractions to the thromboxane mimetic U-46619. Thus ACh and SNP exert delayed inhibition of EDCF-mediated contractions via distinct pathways. The effect of ACh is NO independent and upstream of the increase in calcium concentration that triggers the release of EDCF. The effect of SNP is downstream of the calcium rise and is mainly NO dependent.

The ability of the smooth muscle of the human umbilical artery to relax may vary under physiological and pathological conditions. We investigated the responsiveness of that preparation to relaxation, as well as the influence of reduced oxygen condition on these responses. Rings of human umbilical arteries from full-term Caesarian deliveries were suspended in modified Krebs–Henseleit solutions bubbled with a gas mixture of 95% O 2 : 5% CO 2 (normal oxygen condition) or 2.5% O 2 : 8% CO 2 balanced with N 2 (reduced oxygen condition). These rings were contracted with potassium chloride, serotonin or the thromboxane A 2 mimetic U46619, before being exposed to either the nitrovasodilator sodium nitroprusside, the potassium channel opener levcromakalim or the calcium channel antagonist amlodipine. While sodium nitroprusside elicited relaxation in this blood vessel, the maximal relaxation to the nitric oxide donor was significantly smaller than that induced by levcromakalim and amlodipine. The nature of the constrictor agent used, or changes of oxygen conditions did not significantly affect the relaxation profile of this human blood vessel. These data suggest that the smooth muscle of the human umbilical artery may be less responsive to vasodilators that act via the nitric oxide pathway. Moreover, vascular responses of umbilical arterial smooth muscle to relaxing agents do not alter under hypoxic or different vasoconstricting conditions.

Thiazolidinediones are a new class of anti-diabetic agents which increase insulin sensitivity by binding to the peroxisome proliferator-activated receptor gamma (PPAR(gamma)) and stimulating the expression of insulin-responsive genes involved in glucose and lipid metabolism. These drugs also have vasodilatory and anti-proliferative effects on vascular smooth muscle cells. However the mechanisms for these actions are not fully understood. Adenosine is a vasodilator and a substrate of equilibrative nucleoside transporters (ENT). The present study studied the effects of three thiazolidinediones, troglitazone, pioglitazone and ciglitazone, on ENT1 in the human aortic smooth muscle cells (HASMCs). Although incubating HASMCs for 48h with thiazolidinediones had no effect on ENT1 mRNA and protein levels, troglitazone acutely inhibited [3H]adenosine uptake and [3H]NBMPR binding of HASMCs with IC50 values of 2.35+/-0.35 and 3.99+/-0.57microM, respectively. The effect of troglitazone on ENT1 was PPAR(gamma)-independent and kinetic studies revealed that troglitazone was a competitive inhibitor of ENT1. In contrast, pioglitazone and ciglitazone had minimal effects on [3H]adenosine uptake by HASMCs. Troglitazone differs from pioglitazone and ciglitazone in that its side-chain contains a Vitamin E moiety. The difference in structure of troglitazone did not account for its inhibitory effect on ENT1 because Vitamin E did not inhibit [3H]adenosine uptake by HASMCs. Using the nucleoside transporter deficient PK15NTD cells stably expressing ENT1 and ENT2, it was found that troglitazone inhibited ENT1 but had no effect on ENT2. From these results, it is suggested that troglitazone may enhance the vasodilatory effect of adenosine by inhibiting ENT1. Pharmacologically, troglitazone is a novel inhibitor of ENT1.

Genistein, a phytoestrogen, possesses cardioprotective effects. Responses to genistein (0.1-100 microM) were assessed in 9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F(2 alpha) (U46619)-contracted porcine coronary arterial rings, with significant relaxations at high concentrations. At concentrations with little relaxation, genistein (0.3-3 microM) did not affect relaxation produced by bradykinin and the calcium ionophore, A23187. In contrast, sodium nitroprusside- and cromakalim-induced relaxations were enhanced by genistein (3 microM). N(omega)-nitro-L-arginine methyl ester (L-NAME) (300 microM) or Triton X-100 (0.5%) did not affect the enhancement of relaxation by genistein. The tyrosine kinase inhibitor, tyrphostin 23 (30 microM), had no effect on sodium nitroprusside-elicited relaxation. In summary, genistein relaxed porcine coronary artery at relatively high concentrations. At a physiologically relevant concentration (3 microM), it is devoid of significant vascular effect, but enhanced endothelium-independent relaxations. This effect of genistein does not involve the nitric oxide synthase (NOS) pathway and the endothelium, and is mediated through a mechanism different from tyrosine kinase inhibition.

While alterations in cholesterol and lipoprotein profiles partly account for menopause being a risk factor for coronary heart disease, recent studies have suggested that 17 beta-estradiol may have vascular effects. Our aims were to study the short-term effects of 17 beta-estradiol on vascular function in isolated porcine coronary artery rings. Concomitantly, we sought to determine if physiological concentrations of 17 beta-estradiol could acutely potentiate relaxation17 alpha- and 17 beta-estradiol at pharmacological (1 microM) concentrations produced relaxation in U46619-pre-contracted porcine coronary artery rings. Relaxation evoked by 17 beta-estradiol was not reversed by the estrogen receptor antagonists tamoxifen and ICI 182780. Following 20 min exposure to a physiological concentration of 17 beta-estradiol (1 nM), which on its own had no effect, relaxation elicited by cromakalim, levcromakalim and sodium nitroprusside, but not bradykinin or calcium ionophore A23187, were significantly enhanced. This potentiating action was also insensitive to tamoxifen and ICI 182780. Our data provide evidence for an acute indirect relaxant action of 17 beta-estradiol and suggest that it may be via a tamoxifen- and ICI 182780-insensitive estrogen receptor. While this response was only observed at pharmacological concentrations, the potentiation of cromakalim, levcromakalim and sodium nitroprusside relaxation was evident in the presence of a physiological concentration (1 nM) of 17 beta-estradiol.These results demonstrate that short-term exposure to 17 beta-estradiol, at concentrations that have no effect on their own, can enhance vasorelaxation. These vascular effects may partly account for some of the acute effects of 17 beta-estradiol on blood flow.

In this study, we assessed the effects of O-demethyl encainide (0.5 μM), the most active metabolite of encainide, and the combination with 3-methoxy-O-demethyl encainide (0.5 μM) and encainide (0.1 μM) on cardiac action potential characteristics in normal canine Purkinje fibers and Purkinje fibers surviving 24 h of myocardial ischemia. O-demethyl encainide decreased [Formula: see text] and conduction in normal Purkinje fibers and Purkinje fibers surviving infarction. Further decreases were observed with the combination. Action potential duration at both 50 and 95% repolarization was decreased by O-demethyl encainide. The combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide had no further effect. The combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide produced a smaller change in effective refractory period than O-demethyl encainide in normal Purkinje fibers and in Purkinje fibers surviving infarction. O-demethyl encainide and the combination shifted the membrane responsiveness curve to more negative potentials in both normal Purkinje fibers and Purkinje fibers surviving infarction. It is apparent from this study that there are differences in the effects of O-demethyl encainide and the combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide in normal Purkinje fibers compared with Purkinje fibers surviving infarction. Also, the combination used in this study had different electrophysiological effects than those of O-demethyl encainide alone.Key words: encainide, metabolites, electrophysiological effects, Purkinje fibers, infarction.

The electrophysiological effects of flecainide, 0.1, 0.5 and 1.0 mg/l, on action potential characteristics were examined in normal Purkinje fibers and Purkinje fibers surviving infarction, at stimulation cycle lengths of 1000 and 300 ms. Overshoot, amplitude and dV/dtmax were reduced significantly in a dose-dependent manner by flecainide in both normal Purkinje fibers and Purkinje fibers surviving infarction. Action potential duration was shortened by flecainide to a greater extent: at the cycle length of 1000 ms compared to 300 ms; at 50% repolarization compared to 90%; and in normal preparations compared to infarcted preparations. Activation time was increased by flecainide in a dose-dependent manner in both types of preparations. This effect was enhanced at the shorter cycle length. In both normal and infarcted preparations, flecainide shortened the ERP of those having a longer initial ERP and lengthened those with a shorter ERP. This effect was significant only at the stimulation cycle length of 1000 ms. This study indicates that flecainide has properties unique and different from traditional antiarrhythmic drugs.

Intracellular action potentials were recorded from normal canine Purkinje fibers using the standard microelectrode technique. The effects of lidocaine (2 and 5 mg/1), tocainide (10 and 20 mg/1) and verapamil (0.5 and 1.0 mg/1) on action potential characteristics as well as conduction times were measured at cycle lengths of 1000, 800, 600, 500, 400 and 300 ms. Lidocaine and tocainide shortened APD50 % and APD90 %, and verapamil shortened APD50 % and lengthened APD90 %. All of these effects were greatest at the longest cycle lengths. A similar interaction of changes in ERP with cycle length was observed for lidocaine and tocainide. Verapamil increased ERP but this effect was not significant. Tocainide and verapamil reduced dV/dtmax while lidocaine had no significant effect. All three drugs increased conduction time. This effect was accentuated at shorter cycle lengths. No direct relationship between dV/dtmax and conduction times were observed. These results indicate that many of the electrophysiological effects of lidocaine, tocainide and verapamil are modified by cycle length. We propose that future studies on the electrophysiology of antiarrhythmic drugs should include stimulation both at long and short cycle lengths.

The modulation of rat platelet phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) and rat platelet aggregation by mepacrine was investigated. The 2-acyl specificity of phospholipase A2 activity was confirmed by using 1-[14C]palmitoyl-2-[3H]arachidonylphosphatidylcholine as substrate. Under optimal pH, phospholipase A2 activity was not affected by aspirin but was inhibited by indomethacin. Contrary to previous reports, a biphasic modulatory role of mepacrine on phospholipase A2 activity and platelet aggregation was demonstrated. The data suggest that platelet aggregation is mediated via phospholipase A2.

The effects of 10 and 20 mg/l of tocainide on transmembrane action potential characteristics were examined in Purkinje fibers surviving infraction. Infracted tissue was obtained from canine hearts 24 h after coronary artery ligation. The preparation was stimulated at basic cycle lengths (BCL) of 1000−300 ms. Tocainide reduced the overshoot and amplitude of Purkinje fibers surviving infarction. The maximum upstroke velocity (V max ) was decreased by tocainine in a dose dependent manner. This effect was more prominent at the shorter BCL. Statistical analysis revealed a significant interaction of the BCL with the drug effect on overshoot, amplitude, V max and action potential durations (APD 50% and APD 90% ). Tocainide reduced the effective refractory period (ERP) at the BCL of 1000 ms, but had no significant effect at the BCL of 300 ms. Membrane responsiveness and steady state characteristics of V max were shifted significantly to more negative membrane potentials by tocainide. Investigation of the recovery kinetics of V max in the presence of tocainide showed an exponential recovery of V max with a time constant of 514 ms. These results support the finding that the effect of tocainide on V max and conduction is enhanced at faster rates of stimulation. Thus tocainide may be able to depress conduction to produce bidirectional block in the termination of ventricular tachycardia caused by reentry, while having minimal effect on conduction at normal heart rates.

KHNNAIRD, A. A. A., ad R. Y. K. MAN. 1986. The effect of lidocaine compared with verapamil on the enhanced ventricular rhythm in the infacted canine heart. Can. 9. Physio1. Phmacol. 64: 4'72-476. MyocardiaB ischemia was produced in dogs by the occlusion of the left anterior descending (LAD) coronary artery for 24 or 48 h. After complete atrioventricular block was produced, enhanced ventricular rhy%hm was observed in all animals. The enhanced ventricular rhythm showed multiple QRS configurations and had spontaneous cycle lengths (SCL) of 397 5 18 ms (n = 20) after 24 h of LAD occlusion and 446 2 23 ms (n = 28) after 48 h of LAD occlusion. Overdrive pacing did not result in the termination of the enhanced ventricular rhythm in any experiment. hspranolol, as a cumulative dose of 2.5 - 2.6 mglkg i. v., also did not abolish the enhanced ventricular rhythm. In 24-h infarcted hearts, lidocaine abolished the enhanced ventricular rhythm in 1 sf 1 1 experiments. In the remaining 10 experiments, the ventricular SCL was increased from 401 & 22 to 49 1 & 26 ms after a cumulative dose of 8.8 2 0.7 mg/kg of lidocaine. In the presence of verapamil, given as a cumulative dose of 0.60& 0.1 1 mg/kg, the ventricular SCL was increased from 401 2 33 to 482 + 64 ms (n = 9). In 48-h infacted hearts, lidocaine abolished the enhanced ventricular rhythm in 5 of 1 B experiments. Both lidocaine and verapamil increased the SCL of heats in which the enhanced ventricular rhythm persisted. Analysis of variance showed that only the increase in SCL by lidocaine in 48-h infucted hems was statistically significant. The atrial and idioventricanlar rhythms in noninfmcted hem responded differently to lidocdne and verapamil. The results suggest that some electmphysiolsgical effects of antimhythrnic dmgs in the no1ma8 heart may not be applicable to those in the diseased situation. KINNAIHPD, A. A. A., et W. Y. K. MAN, 1986. The effect of Bidocaine compared with verapamil on the enhanced ventricular