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Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75–96%), and the isolated yields were also good (64–87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. 13 C NMR spectra showed spin–spin coupling between 13 C and 15 N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] Increase in MARCKS phosphorylation was observed in SH-SY5Y cells by MeHg. [•] MARCKS knock-down facilitated loss of cell viability induced by MeHg. [•] Augmentation of MARCKS phosphorylation was observed in MeHg-treated rats. [Copyright &y& Elsevier]

Abstract: Aims: Hypersensitivity of platelets due to increased platelet cholesterol levels has been reported in hypercholesterolemia. However, the signaling pathways linking increased platelet reactivity and cholesterol contents are not fully understood. This study aims to determine the direct effect of cholesterol enrichment of platelets on the pathways including Ca2+ mobilization and secondary feedback agonists such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Main methods: In vitro cholesterol enrichment of rabbit platelets was performed by incubation with cholesterol complexed with methyl-β-cyclodextrin. Ca2+ mobilization was monitored using platelets loaded with fura-PE3/AM, a fluorescent calcium indicator. Released ATP and TXB2 from platelets were measured by a luciferin–luciferase ATP assay system and a TXB2 ELISA Kit, respectively. Key findings: Cholesterol enrichment of rabbit platelets significantly enhanced Ca2+ mobilization induced by thrombin, accompanying an augmented Ca2+ entry. The augmentation of Ca2+ entry by cholesterol enrichment was significantly suppressed by treatment with inhibitors for secondary feedback agonists. In cholesterol-enriched platelets, the amount of released ATP or TXB2 induced by thrombin was not significantly altered in comparison with control platelets, whereas an increase in [Ca2+]i induced by ADP or U46619, a TXA2 mimetic, was significantly enhanced. Significance: These results suggest that cholesterol enrichment of rabbit platelets results in enhanced Ca2+ mobilization via ADP/TXA2-dependent augmentation of the Ca2+ entry pathway. The results reveal a novel mechanism by which platelet hypersensitivity is regulated by cholesterol contents. [Copyright &y& Elsevier]

Aims Methylmercury (MeHg) exposure results in increased risk of hypertension and cardiovascular diseases. In this study, we aimed to investigate whether the in vivo exposure of MeHg in mice affects blood pressure and basilar arterial responses to angiotensin II (Ang II) and acetylcholine (ACh), which are important modulators of cerebrovascular autoregulation. Main methods Mice were exposed to MeHg (40 ppm) in drinking water for 21 days. Blood pressure was measured in conscious mice by an indirect tail-cuff method. Functional studies of the isolated arteries' response to vasoactive substances were performed using a micro-organ-bath system. Key findings Systolic and mean blood pressures were significantly increased after 2 and 3 weeks of treatment with MeHg, respectively. Ang II-induced contraction in an isolated basilar artery, which is mediated via Rho-kinase activation, was increased in MeHg-treated mice. ACh-induced relaxation, which is dependent on NO production from the endothelial cells, was decreased in MeHg-treated mice. However, alterations of vascular responses to Ang II and ACh were not observed in the isolated thoracic aorta. Significance This study demonstrated that the cerebral vasculature appears to be particularly sensitive to in vivo exposure of MeHg. Our results suggest that in vivo MeHg increases blood pressure and causes alterations in the cerebrovascular reactivity in response to Ang II and ACh through enhancement of Rho-kinase activity and inhibition of NO bioavailability, respectively. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the histamine responsiveness of basilar arterial rings isolated from chicken. We also examined whether endothelial cells were involved in the histamine responsiveness and in resting vascular tone. Histamine induced concentration-dependent relaxations under condition of precontraction by 5-hydroxytryptamine. The concentration–response curve for histamine was shifted to the right by diphenhydramine (a H1 receptor antagonist), cimetidine (a H2 receptor antagonist) and Nω-nitro-l-arginine (l-NNA, a nitric oxide synthase inhibitor); however, indomethacin (a cyclooxygenase inhibitor) had no significant effect on it. Treatment with l-NNA shifted the concentration–response curve of histamine to the right in the presence of cimetidine, but not in the presence of diphenhydramine. Treatment with cimetidine shifted the concentration–response curve of histamine to the right in the presence of diphenhydramine. l-NNA induced a contraction but indomethacin had no effect on the resting vascular tone. These results suggest that histamine-induced relaxation is mediated via activation of H1 receptors located on endothelial cells and H2 receptors located on smooth muscle cells. The main relaxing factor released from endothelial cells is probably nitric oxide. The resting vascular tone was modulated by spontaneously released nitric oxide, but not by prostaglandins or thromboxane A2. [Copyright &y& Elsevier]

The response of basilar arteries to noradrenaline varies among many animal species, but remains little studied in poultry. Accordingly, we aimed to characterize the adrenergic receptor (AR) subtypes that modulate vascular response in basilar arteries in the chicken, with isometric recording of arterial ring tension using an organ bath. We demonstrated the presence of both alpha and beta (α and β) receptor subtypes through evaluating the response to noradrenaline, with and without a range of β-AR and α-AR antagonists. The concentration-dependent relaxations then induced by a range of β-AR agonists indicated a potency ranking of isoproterenol > noradrenaline > adrenaline > procaterol. We then investigated the effects of β-AR antagonists that attenuate the effect of isoproterenol (propranolol for β 1,2,3 -ARs, atenolol for β 1 -ARs, butoxamine for β 2 -ARs, and SR 59230A for β 3 -ARs), with Schild regression analysis, ascertaining multiple β-AR subtypes, with neither the β 1 -AR nor the β 2 -AR as the dominant subtype. SR 59230A was the only antagonist to yield a pA 2 value (7.52) close to the reported equivalent for the relevant receptor subtype. Furthermore, treatment with SR 58611 (a β 3 -AR agonist) induced relaxation, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Additionally, treating basilar arterial strips (containing endothelium) with SR 58611 induced nitric oxide (NO) production, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Based on this first characterization of AR subtypes in chicken basilar arteries (to our knowledge), we suggest that α- and β-ARs are involved in contraction and relaxation, and that β 3 -ARs, especially those on the endothelium, may play an important role in vasodilation via NO release. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance In Vietnamese traditional herbalism, there are conflicting opinions about the effect of Artemisia vulgaris L. (AVL, English name: mugwort) on hypertension. Some ethnic doctors recommend the use of AVL for treatment of hypertension, whereas others advise against it. The purpose of this study was to clarify the pharmacological characteristics of AVL in isolated arteries to explain the conflicts surrounding the use of AVL for treatment of hypertension. Materials and methods We initially performed a functional study using an organ bath system to investigate the effect of AVL extract on isolated porcine basilar artery. We then measured the change in intracellular free Ca 2+ concentration elicited by AVL using cultured smooth muscle cells loaded with the Ca 2+ indicator fluo-4. Finally, using HPLC, we determined the active components in AVL. Results and discussion AVL induced vasoconstriction at resting tension, and endothelial removal enhanced this effect significantly. Pretreatment with PD123319 (an AT 2 receptor antagonist), Nω - nitro - L - arginine (a nitric oxide synthase inhibitor), or both, also enhanced this effect. AVL-induced contraction was competitively inhibited by methiothepin (a 5-HT 1 and 5-HT 2 receptor antagonist) in the presence of ketanserin (a 5-HT 2 receptor antagonist). Removal of extracellular calcium with nifedipine (an L-type Ca 2+ channel blocker) or ruthenium red (a ryanodine receptor blocker) significantly reduced AVL-induced contraction, whereas losartan (an AT 1 receptor antagonist) and diphenhydramine (a H 1 receptor antagonist) had no effect on this contraction. AVL increased the intracellular free Ca 2+ concentration in cultured cells, and this increment was inhibited by methiothepin. HPLC analysis revealed that the retention time of the first peak in the AVL profile was similar to that of the 5-HT standard, and that addition of 5-HT to the AVL sample enhanced this peak. On the other hand, AVL induced endothelium-independent relaxation under precontracted conditions with 60 mM KCl. Captopril (an angiotensin converting enzyme inhibitor), atenolol (a β 1 receptor antagonist) and cimetidine (a H 2 receptor antagonist) had no effect on this relaxation. In Ca 2+ -free 60 mM KCl-containing solution, pretreatment with AVL significantly inhibited CaCl 2 -induced contraction. Conclusion For the first time, the present study has demonstrated that AVL has two opposite effects, contraction and relaxation, on isolated artery, which may help to explain the conflicting indications for AVL in traditional herbalism. 5-HT is a significant factor affecting artery contraction in the presence of AVL. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: The wax gourd (Benincasa hispida (Thunb) Cong.) is a long-season vegetable that has been used in traditional Chinese medicine to treat high blood pressure. However, precise details of its effect and the mechanism of action involved are still lacking. Materials and methods: Ten-fold-condensed wax gourd juice was used for the experiments. We measured (1) blood pressure of anesthetized normal Wistar rats in vivo, (2) isolated rat aortic contraction and relaxation, and (3) nitric oxide production from cultured porcine endothelial cells. The rats mentioned had not been treated with the investigational medicine. Results: Intravenous injection of the juice produced a dose-dependent decrease in blood pressure. Treatment with the juice induced concentration-dependent relaxation of isolated rat aortic rings that had been precontracted with noradrenaline. The relaxation induced by the juice was strongly inhibited by treatment with the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester hydrochloride (l-NAME) or endothelial denudation. Treatment with the juice produced NO from cultured porcine aortic endothelial cells. This NO production was significantly inhibited by l-NAME. Conclusions: The present findings suggest that wax gourd juice exerts a hypotensive effect via endothelium-dependent vasodilation. The main endothelium-derived relaxing factor involved might be NO. [Copyright &y& Elsevier]

The responsiveness of the basilar artery to intrinsic vasoactive substances is species-specific and can be a unique characteristic. We investigated the responsiveness of the bat (Rhinolophus ferrumequinum) basilar artery to noradrenaline (NA), 5-hydroxytryptamine (5-HT), angiotensin (Ang) II, bradykinin (BK), histamine (His), and acetylcholine (ACh). NA, 5-HT, Ang II, and BK induced contraction, whereas His and ACh induced relaxation, in a concentration-dependent manner. The NA cumulative concentration–response curve was shifted to the right in parallel with phentolamine (an α-antagonist). However, propranolol, a β-antagonist, had no significant effect. The 5-HT curve was shifted to the right in parallel by ketanserin (a 5-HT 2 antagonist) and methiothepin (a 5-HT 1 and 5-HT 2 antagonist). Losartan (an AT 1 antagonist) shifted the Ang II curve to the right, whereas PD123319 (an AT 2 antagonist) had no significant effect. L-NA, indomethacin, and des-Arg9-[Leu8]-BK (a B 1 antagonist) did not significantly affect BK-induced contractions. HOE140 (a B 2 antagonist) shifted the BK concentration–response curve to the right. The His curve was shifted to the right weakly by diphenhydramine (an H 1 antagonist) and strongly by cimetidine (a H 2 antagonist). ACh-induced relaxation was significantly inhibited by L-NA, atropine, and pFHHSiD (a muscarinic M 3 antagonist), whereas pirenzepine and methoctramine (muscarinic M 1 and M 2 antagonists, respectively) showed no significant effects. At a resting vascular tone, L-NA-induced contraction and indomethacin induced relaxation. These results suggest that α-adrenergic, 5-HT 1 , 5-HT 2 , AT 1 , and B 2 receptors might be important in arterial contraction, whereas M 3 and H 2 (>H 1) receptors might modify these contractions, inducing relaxation. [Display omitted] • NA, 5-HT, Ang II, and BK induced contraction in a concentration-dependent manner. • His and ACh induced relaxation in a concentration-dependent manner. • α-Adrenergic, 5-HT 1 , 5-HT 2 , AT 1 , and B 2 receptors might be involved in NA, 5-HT, Ang II, and BK-induced contraction, respectively. • M 3 and H 2 (>H 1) receptors might modify these contractions to relaxations. • Bat basilar arterial responses to these substances are close to those of horses. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the responsiveness of basilar arterial rings isolated from snakes to noradrenaline (NA), acetylcholine (ACh), histamine (His), 5-hydroxytryptamine (5-HT), mammalian bradykinin (BK) and rattlesnake BK. We also examined whether endothelial cells were involved in the responsiveness to ACh, BK, rattlesnake BK and in their resting vascular tone. NA and 5-HT induced concentration-dependent contractions. The cumulative concentration response curves of NA and 5-HT were shifted to the right in parallel by phentolamine (an α antagonist) and methiothepin (a 5-HT1 and 5-HT2 antagonist), respectively. However, ketanserin (a 5-HT2 antagonist) had no effect on the cumulative concentration response curve of 5-HT. His, ACh, BK and rattlesnake BK had no effect on resting vascular tone; however, rattlesnake BK and sodium nitroprusside relaxed arteries precontracted by 5-HT. The rattlesnake BK-induced relaxations were almost abolished by L-nitro arginine (L-NA, a nitric oxide synthase inhibitor). L-NA and indomethacin (a cyclooxygenase inhibitor) had no effect on resting vascular tone or on precontracted arteries. These results suggest that α and 5-HT1 receptor subtypes might be important in arterial contraction. Endothelial cells might play an important role in the responsiveness of snake basilar arteries to rattlesnake BK, but they might not be involved in the responsiveness to ACh, BK and in resting vascular tone. [Copyright &y& Elsevier]