Endothelium-intact Rings Research Articles

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

Aim To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. Methods The relaxation response to NaOH-induced extracellular alkalinization (7.4–8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10 −6 M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10 −5 M), NG-nitro- l-arginine methyl ester (L-NAME, 10 −4 M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10 −7 M), 2,5-dimethylbenzimidazole (DMB, 2 × 10 −5 M) and methyl-β-cyclodextrin (10 −2 M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2 × 10 −5 M). Results The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca 2+/calmodulin and Na +/Ca 2+ exchanger (NCX), and partially blunted by the caveolae disassembly. Conclusions These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca 2+ concentration and activating the Ca 2+/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.

Vascular relaxation of canine visceral arteries after ischemia by means of supraceliac aortic cross-clamping followed by reperfusion

BackgroundThe supraceliac aortic cross-clamping can be an option to save patients with hipovolemic shock due to abdominal trauma. However, this maneuver is associated with ischemia/reperfusion (I/R) injury strongly related to oxidative stress and reduction of nitric oxide bioavailability. Moreover, several studies demonstrated impairment in relaxation after I/R, but the time course of I/R necessary to induce vascular dysfunction is still controversial. We investigated whether 60 minutes of ischemia followed by 30 minutes of reperfusion do not change the relaxation of visceral arteries nor the plasma and renal levels of malondialdehyde (MDA) and nitrite plus nitrate (NOx).MethodsMale mongrel dogs (n = 27) were randomly allocated in one of the three groups: sham (no clamping, n = 9), ischemia (supraceliac aortic cross-clamping for 60 minutes, n = 9), and I/R (60 minutes of ischemia followed by reperfusion for 30 minutes, n = 9). Relaxation of visceral arteries (celiac trunk, renal and superior mesenteric arteries) was studied in organ chambers. MDA and NOx concentrations were determined using a commercially available kit and an ozone-based chemiluminescence assay, respectively.ResultsBoth acetylcholine and calcium ionophore caused relaxation in endothelium-intact rings and no statistical differences were observed among the three groups. Sodium nitroprusside promoted relaxation in endothelium-denuded rings, and there were no inter-group statistical differences. Both plasma and renal concentrations of MDA and NOx showed no significant difference among the groups.ConclusionSupraceliac aortic cross-clamping for 60 minutes alone and followed by 30 minutes of reperfusion did not impair relaxation of canine visceral arteries nor evoke biochemical alterations in plasma or renal tissue.

Vascular dilation by paeonol — A mechanism study

The goal of this study was to investigate the mechanism underlaying the vasodilatory effect of paeonol, a major active element from the root bark of Chinese herbs Paeonia suffruticosa Andr. and Cynanchum paniculatum (Bunge) Kitagawa. Paeonol relaxed isolated rat aorta rings by 95.6% while the 10−6M forskolin-induced vasodilatation used as 100%. The EC50 of vasodilatation by paeonol is 2.9×10−4M. Although paeonol exerted endothelium-independent relaxation, L-NAME treatment inhibited paeonol-induced vasodilation of endothelium intact rings, while indomethacin did not. Both L-NAME and ODQ did not affect paeonol relaxation in the rings without endothelium. In addition, paeonol markedly elevated NO generation in cultured endothelial cells. Pre-treatment of propranolol, glibenclamide, TEA and BaCl2 did not affect paeonol relaxation of endothelium removed rings. On the other hand, pre-treated of rings (without endothelium) with paeonol markedly blocked vasoconstriction induced by AngII, PGF2α, 5-HT, dopamine, vasopressin, endothelin-1 and PE. The paeonol incubation also significantly attenuated KCl-induced contraction which mainly depended on Ca2+ influx. In Ca2+-free medium (containing 10−4M of EGTA and 60mM of KCl), paeonol suppressed the contraction curve of CaCl2. In addition, paeonol also inhibited contraction by PE in Ca2+ free solution (containing 10−4M of EGTA) which mainly relied on intracellular Ca2+ release. Whole-cell patch-clamp experiment showed that paeonol shifted the I–V curve and the peak value of calcium currents was significantly inhibited. In conclusion, our study suggested that voltage-dependent and receptor-operated Ca2+ channel, as well as intracellular Ca2+ release were all inhibited by paeonol. An intracellular Ca2+ regulatory mechanism may be responsible to potent vasodilatory effect of paeonol.

Endothelium-dependent and -independent coronary relaxation induced by urocortin.

Urocortin, a newly identified polypeptide, possesses cardiac effects. However, the underlying mechanism of its coronary action is still unclear. In the present study we investigated the possible role of endothelial nitric oxide and Ba2+-sensitive K+ channels in the endothelium-dependent relaxant response to urocortin in the isolated rat left anterior descending coronary arteries. Changes of vessel tone were measured in microvessel myographs. Urocortin produced both endothelium-dependent and -independent relaxation with IC50 of 2.52 nM and 16.5 nM, respectively. Denuation of endothelium decreased the relaxing potency of urocortin. In the endothelium-intact rings pretreated with 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME) or 10 microM 1H-[1,2,4]oxadiazolo[4,2-alpha]quinoxalin-1-one (ODQ), the urocortin-induced relaxation was similar to that observed in endothelium-denuded rings. The relaxant response to urocortin was markedly reduced in endothelium-intact rings preconstricted by 35 mM K+. Pretreatment with 100 microM BaCl2 significantly reduced urocortin-induced relaxation without an effect on the maximum relaxation. Combined treatment with BaCl2 plus L-NAME did not produce additive inhibition. In contrast, BaCl2 did not alter urocortin-induced relaxation in the endothelium-denuded rings. In the endothelium-denuded rings, BaCl2 at 100 microM also inhibited nitric oxide donor-induced relaxation. In conclusion, our results suggest that urocortin-induced endothelium-dependent relaxation of rat coronary arteries is primarily mediated by endothelial nitric oxide and subsequent activation of Ba2+-sensitive K+ channels. The urocortin-induced endothelium-dependent relaxation appears to be cyclic GMP-dependent.

Mechanisms involved in the effects of endothelin-1 in pig prostatic small arteries

Since endothelin-1 (ET-1) is involved in prostatic disorders, the current study investigated the mechanisms underlying the ET-1-induced effects in pig prostatic small arteries. The experiments were performed in rings mounted in microvascular myographs containing physiological saline solution at 37 oC for isometric force recordings. On basal tension, ET-1 (0.1–30 nM) evoked concentration-dependent contractions, which were enhanced by endothelium removal. ET-1 contractions were inhibited by blockade of endothelin ET A and ET B receptors, extracellular Ca 2+ removal and blockade of voltage-dependent (L-type)- and non-voltage-dependent-Ca 2+ channels. On endothelium intact rings precontracted with noradrenaline, the ET B endothelin receptor agonist BQ3020 promoted a concentration-dependent relaxation which was reduced by blockade of ET B receptors, nitric oxide synthase, guanylyl cyclase and prostanoids synthesis. Endothelium removal abolished its relaxant response and unmasked a BQ3020-induced contraction. Tetraethylammonium and 4-aminopyridine, blockers of non-selective K + channels and voltage-dependent K + (Kv) channels, respectively, inhibited the relaxations to BQ3020. Iberiotoxin, apamin and glibenclamide, blockers of large and small Ca 2+-activated- and ATP-dependent- K + channels, respectively, failed to modify these responses. These data suggest that ET-1 promotes contraction of pig prostatic small arteries by activating vascular smooth muscle contractile endothelin ET A and ET B receptors coupled to extracellular Ca 2+ entry, via voltage-dependent (L-type)- and non-voltage-dependent Ca 2+ channels, also being due to intracellular Ca 2+ mobilization. In addition, a population of endothelial ET B receptors mediates vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and Kv channels.

Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery

It is known that long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have beneficial effects on cardiovascular function including pulmonary hypertension. The purpose of the present study was to examine the mechanisms involved in EPA-induced relaxation of sheep isolated pulmonary artery by measuring isometric tension. Nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) was measured by Greiss method in the presence of the inducible nitric oxide synthase (iNOS) selective inhibitor N-[[3-(aminomethyl) phenyl]methyl]-ethanimidamide, dihydrochloride (1400 W). EPA (10(-)(7)-10(-)(4)M) caused concentration-dependent relaxation of sheep pulmonary artery with a pD(2) of 5.56+/-0.09 and E(max) of 87.40+/-3.10% (n=9). N(G)-nitro-L-arginine methyl ester (L-NAME) 100 microM significantly attenuated (E(max) 41.95+/-6.70%; n=8) EPA-induced relaxation of endothelium intact arterial rings. Similarly, endothelium denudation markedly inhibited (E(max) 17.60+/-1.21%; n=4) EPA-induced relaxation. EPA (30 microM) significantly increased the cNOS-derived NO release (10.17+/-0.96; n=8 versus control 7.43+/-0.78 pmol/mg tissue wet wt./h; n=7) in endothelium intact vessels. However, EPA-stimulated NO release was markedly blunted by either 100 microM L-NAME (7.07+/-0.54 pmol/mg tissue wet wt./h; n=8) or endothelium removal (6.97+/-0.87 pmol/mg tissue wet wt./h; n=17). In endothelium-denuded K(+) (80 mM)-depolarized arterial rings, EPA (30 microM) significantly inhibited CaCl(2)-induced contractions (E(max) 42.77+/-5.90% versus control 94.78+/-9.82%; n=5). The fatty acid also inhibited nifedipine (1 microM)-insensitive 5-HT-induced contractions in this vessel (E(max) 70.57+/-4.88% versus control 161.50+/-17.46%; n=5). In conclusion, EPA relaxes sheep pulmonary artery primarily through endothelium-dependent NO release, and the residual endothelium-independent relaxation may result from inhibition of Ca(2+)-influx through L-type calcium channels, as well as 5-HT-stimulated intracellular Ca(2+) release.

The Direct Effect of Levobupivacaine in Isolated Rat Aorta Involves Lipoxygenase Pathway Activation and Endothelial Nitric Oxide Release

Levobupivacaine is a long-acting local anesthetic with a clinical profile similar to that of racemic bupivacaine but with a greater margin of safety. Levobupivacaine produces dose-dependent vasoconstriction in vivo. Our goal in this in vitro study was to investigate the role of pathways involved in arachidonic acid metabolism in the levobupivacaine-induced contraction of isolated rat aorta and to determine which endothelium-derived vasodilators are involved in the modulation of levobupivacaine-induced contraction. Rat thoracic aortic rings were isolated and suspended for isometric tension recording. Cumulative levobupivacaine dose-response curves over a range of 10(-6) to 3 x 10(-4) M were constructed in 1) aortic rings with no drug pretreatment; 2) endothelium-denuded rings pretreated with quinacrine dihydrochloride (nonspecific phospholipase A(2) inhibitor: 2 x 10(-5), 4 x 10(-5) M), nordihydroguaiaretic acid (NDGA) (lipoxygenase inhibitor: 10(-5), 3 x 10(-5) M), indomethacin (nonspecific cyclooxygenase inhibitor: 10(-5) M), AA-861 (5-lipoxygenase inhibitor: 10(-5), 5 x 10(-5) M), fluconazole (cytochrome P450 epoxygenase inhibitor: 10(-5) M), verapamil (10(-5) M), or calcium-free solution; and 3) endothelium-intact rings pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor: 5 x 10(-5) M), indomethacin, or fluconazole. Levobupivacaine-induced contractile response at each concentration (10(-4), 3 x 10(-4) M) was assessed in endothelium-denuded rings. Dose-response curves for potassium chloride in endothelium-denuded rings were generated in the presence or absence of NDGA and AA-861. Intracellular Ca(2+) levels were monitored by Ca(2+) image analysis using Fluo-4 fluorescence in vascular smooth muscle cells treated with levobupivacaine alone or AA-861 plus levobupivacaine. Levobupivacaine produced a tonic contraction in isolated rat aorta rings; this response was maximal at 10(-4) M levobupivacaine and gradually attenuated at 3 x 10(-4) M levobupivacaine. Levobupivacaine-induced contractions of endothelium-denuded rings were larger than those of endothelium-intact rings. Levobupivacaine-induced contraction of endothelium-denuded rings was attenuated by quinacrine dihydrochloride, NDGA, AA-861, verapamil, and calcium-free solution and, to a lesser extent, by indomethacin. L-NAME enhanced levobupivacaine-induced contraction of endothelium-intact rings and indomethacin slightly attenuated this contraction. NDGA and AA-861 attenuated the potassium chloride-induced contraction. AA-861 attenuated the levobupivacaine-induced intracellular calcium increase in vascular smooth muscle cells. Our data indicate that levobupivacaine-induced contraction of rat aortic smooth muscle is mediated mainly by activation of the lipoxygenase pathway and in part by activation of the cyclooxygenase pathway. In addition, activation of the lipoxygenase pathway seems to facilitate calcium influx via L-type calcium channels. Endothelial nitric oxide attenuates levobupivacaine-induced contraction.

Altered Endothelium-Dependent Vasoreactivity of Aortic Rings Follows L-Arginine and Potassium Co- Supplementation in Rats

Purpose: The use of L-arginine and potassium chloride separately as supplements has been reported to result in altered vascular reactivity. The concentration of either agent used has varied widely and there has been no report on the outcome of combined supplementation with both agents on vascular reactivity. We therefore designed this study to measure thoracic aortic ring reactivity after five-week co-supplementation with 1% L-arginine and 0.75% potassium chloride.Methods: Endothelium-intact and endothelium-denuded aortic rings obtained from Sprague-Dawley rats which received the combined supplements were subjected to graded concentrations of carbachol or noradrenaline in organ baths. The maximum responses (Emax)and the negative logarithm of the concentration producing 50% of maximum responses (pD2) were computed as indices of vasoreactivity.Results: Co-supplementation significantly enhanced the relaxant effects of carbachol irrespective of the status of the endothelium although relaxation was by far higher in endothelium-intact rings. In concentration-response curves obtained with noradrenaline, Emax and pD2 values were significantly (P < 0.05) reduced in endothelium-intact rings obtainedfrom rats which received the supplements. In endothelium-denuded rings, responses to noradrenaline were not significantly different from those of control.Conclusion: Co-supplementation with the agents enhances the relaxant effect of carbachol independent of the status of the endothelium while attenuating responses to noradrenaline in endothelium-dependent manner. This suggests that oral L-arginine and potassium cosupplementation may possess beneficial vascular effects.

P3. Postganglionic neurones and effectors: P3.1 Vasodilatation induced by nitric oxide donors is potentiated in endothelium intact rat aortic rings

P3. Postganglionic neurones and effectors: P3.1 Vasodilatation induced by nitric oxide donors is potentiated in endothelium intact rat aortic rings

Echinacoside Elicits Endothelium-Dependent Relaxation in Rat Aortic Rings via an NO-cGMP Pathway

The aim of this study was to identify and elucidate the vasorelaxant activity of echinacoside, a phenylethanoid glycoside isolated from the medicinal herb Cistanche tubulosa, and its possible underlying mechanism on isolated rat thoracic aortic rings pre-contracted with phenylephrine (PE, 1 microM) and KCl (60 mM). Echinacoside (30-300 microM) exhibited an acute relaxation in endothelium-intact rings in a concentration-dependent manner, while this relaxation was significantly inhibited in endothelium-denuded condition and in the presence of the endothelial nitric oxide synthase (eNOS) inhibitor, N(W)-nitro-L-arginine methyl ester (L-NNA, 100 microM), an unselective soluble guanylate cyclase blocker, methylene blue (10 microM), the selective sGC inhibitor 1 H-[1, 2, 4]oxadiazolo[4,3- A]quinoxalin-1-one (ODQ, 1 microM); in addition, atropine (1 microM), a selective muscarinic receptor antagonist, partially affected the relaxation. However, the cyclooxygenase inhibitor indomethacin (5 microM) had no influence on the action. Echinacoside enhanced the cyclic guanosine monophosphate (cGMP) production in aortic rings contracted with PE. These results indicate for the first time that echinacoside mediates the endothelium-dependent vasodilator action in rat thoracic aortic rings through nitric oxide (NO)-cGMP pathway.

Thromboxane prostanoid receptor activation impairs endothelial nitric oxide-dependent vasorelaxations: The role of Rho kinase

Activation of thromboxane prostanoid (TP) receptors causes potent vasoconstriction, which contributes to increased vascular tone and blood pressure. The present study examined the hypothesis that stimulation of TP receptor impaired endothelial nitric oxide-mediated vasorelaxation via a Rho kinase-dependent mechanism. The common carotid arteries of Sprague–Dawley rats were isolated and suspended in myograph for measurement of changes in isometric tension. The production of nitric oxide in primary cultured aortic endothelial cells was assayed with an imaging technique and phosphorylated levels of endothelial NOS were determined by Western blot analysis. 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F 2α (U46619) inhibited isoprenaline-induced relaxations in rings with or without endothelium. Treatment with Rho kinase inhibitors, Y27632 (2 μM) or HA 1077 (10 μM) prevented the effect of U46619 only in rings with endothelium while protein kinase C inhibitors were without effect. Rho kinase inhibitors did not affect isoprenaline-induced relaxations in endothelium-intact rings treated with L-NAME or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). Isoprenaline stimulated rises in nitric oxide (NO) production in cultured rat endothelial cells. The increased NO production was inhibited by U46619 (100 nM) and this effect was prevented by treatment with Y27632 but unaffected by the absence of extracellular calcium ions. U46619 attenuated isoprenaline-stimulated phosphorylation of eNOS, which was sensitive to inhibition by Y27632 and HA 1077. U46619-mediated effects were abolished by TP receptor antagonist, S18886 and the TP receptor was present in endothelial cells. The present results demonstrate that Rho kinase activation is likely to be the primary mechanism that underlies the U46619-stimulated TP-receptor-mediated inhibition of endothelial NO production and subsequent endothelium-dependent relaxations to isoprenaline.

Chronic epigallocatechin-gallate improves aortic reactivity of diabetic rats: Underlying mechanisms

Diabetes mellitus is associated with major cardiovascular risk factors which are responsible for excess morbidity and mortality. Green tea catechins including epigallocatechin-3-gallate (EGCG) could exert beneficial health effects to ameliorate cardiovascular and metabolic diseases. Thus, the effect of chronic administration of EGCG was studied on aortic reactivity of streptozotocin (STZ)-diabetic rats. Male diabetic rats received EGCG 25 mg/kg/day for 8 weeks 1 week after diabetes induction. Contractile responses to KCl and phenylephrine (PE) and relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained from aortic rings. Maximum contractile response of endothelium-intact rings to KCL and PE was significantly lower in EGCG-treated diabetic rats relative to untreated diabetic ones. Endothelium removal abolished the significant difference between EGCG-treated and untreated diabetic groups regarding contractile response to KCl and PE. Meanwhile, endothelium-dependent relaxation to ACh was significantly higher in EGCG-treated diabetic rats as compared to diabetic ones. Pretreatment of rings with N(omega)-L-arginine methyl ester (L-NAME) and indomethacin (INDO) significantly attenuated the observed responses. Meanwhile, two-month diabetes resulted in an elevation of malondialdehyde (MDA) and decreased superoxide dismutase (SOD) activity in aortic tissue and EGCG treatment attenuated the increased MDA content and reduced activity of SOD. Therefore, chronic treatment of diabetic rats with EGCG could prevent the abnormal functional changes in vascular reactivity in diabetic rats through nitric oxide- and prostaglandin-dependent pathways and via attenuation of aortic lipid peroxidation.

Extracellular acidosis promote endothelium‐independent and nitric oxide‐dependent vasodilation

Extracellular acidosis (EA) has profound effects on vascular homeostasis since EA‐induced tone alterations are specific to vascular bed. In the present study, we examined the in vitro ("organ chambers") effects of acidosis on dilatory responses of isolated rat thoracic aorta. pH‐response curves (7.4 to 6.5) were generated using HCl (1M) in phenylephrine pre‐contracted endothelium intact and denuded rings. The acidosis pH‐response curves were obtained by gradual addition of HCl to the preparation. The curves were performed with and without tetraethylamonium (TEA), NG‐nitro‐L‐arginina metil ester (L‐NAME) and/or indomethacin (Indo). Relaxation mediated by HCl was increased in endothelium intact rings (73.12±3.66) compared to denuded rings (39.56±3.78) during mild (pH 7.0), but not severe acidosis (pH 6.5) (94.33±1.96 to 91.42±0.95). Mild acidosis‐dilatory effect in endothelium intact rings was markedly reduced by L‐NAME (32.54±5.38) and L‐NAME + Indo (22.80±7.36). In denuded rings, relaxation was reduced only by L‐NAME in mild (11.52±4.18) and in severe acidosis (53.32±6.44). TEA and Indo failed to alter the relaxing effect of acidosis. These results point that acidosis promoted nitric oxide (NO)‐mediated relaxation in rat aorta. Moreover, other factors, besides NO, can be involved in relaxation mediated by EA. Support: FAPESP; FAEPA‐HC/FMRP.

Relaxing Effects of Phytoestrogen α-Zearalanol on Rat Thoracic Aorta Rings in Vitro

The aim of this research is to investigate the vasorelaxing effects and mechanisms involved in the phytoestrogen alpha-zearalanol (alpha-ZAL) in rat thoracic aortas rings. Intact or endothelium denuded rat thoracic aortas rings were put in individual organ chamber to observe the endothelium-dependent or independent vasorelaxing effects of alpha-ZAL (10(-10)-10(-5) M). The thoracic aortas rings were pre-contracted with phenylephrine. The relaxing effects of alpha-ZAL were observed and the influence of N(omega)-nitro-L-arginine methylester (L-NAME, NOS inhibitor), methylene blue (MB, guanylate cyclase inhibitor), charybdotoxin (ChTX, Ca(2+)-activated K+ channel blocker), glibenclamide (ATP-sensitive K+ channel blocker), (-) BayK8644 (L-type Ca2+ channel agonist) and ICI182,780 (estrogen receptor antagonist) were pre-incubated with alpha-ZAL, respectively, to explore the possible mechanisms involved in this vasorelaxation. Furthermore, the Phospho-eNOS expression and cGMP level in the aortas tissue were detected by Western blot and radioimmunity, respectively; the NO level in perfusate was assaied by chromatometry. Our result showed that alpha-ZAL (10(-10)-10(-5) M) induced both endothelium-dependent and -independent relaxation of rat thoracic aortas rings. The vasorelaxing effects of alpha-ZAL were dose-dependent whether the endothelium was intact or not. In endothelium-intact aortas rings, alpha-ZAL-induced vasorelaxation might be inhibited by L-NAME, MB, charybdotoxin, glibenclamide and (-) BayK8644, but not ICI182,780. (-) BayK8644 could also inhibit alpha-ZAL-induced vasorelaxation in endothelium-denuded aortas rings.10(-7)-10(-5) M alpha-ZAL might induce the Phospho-eNOS expression in thoracic aorta tissue, increase the NO level in perfusate and cGMP content in thoracic aorta tissue. Meanwhile, L-NAME might decrease both NO and its downstream cGMP level. Methylene blue might decrease the level of cGMP. These results suggest that alpha-ZAL induces a partly endothelium-dependent relaxation of rat thoracic aortas rings; the possible mechanisms involved in this rapid vasorelaxation include activation of eNOS/NO/cGMP pathway, opening of VSMCs ATP-sensitive and Ca(2+)-activated K+ channels through secretion of EDHF from endothelium. Furthermore, this relaxation also appears to be mediated by both direct and indirect inhibition of voltage-dependent Ca2+ channel of VSMCs, while it is not concerned with activation of estrogen receptor.

Enhanced angiotensin II‐induced aortic constriction in maternally separated rats is endothelium‐dependent and reactive oxygen species (ROS)‐independent.

We previously have reported that Angiotensin II (AngII)‐induced constriction is enhanced in rats subjected to Maternal Separation (MS), a model of early life stress. The aim of this study was to investigate whether NO and/or ROS are involved in the increased Ang II‐induced constriction in MS rats. Male Wistar Kyoto rats were separated from their mother 3 hs/day from day 2 to 14 of life. Unseparated rats (MU) were used as control. Dose‐response curves to AngII (10−12 to 10−4.5 mol/L) were performed in endothelium‐intact and denuded thoracic aortic rings from MS and MU adult rats. NO basal tone was determined using a single dose of PE (10−8M, ±L‐NAME 100 μM). Inhibition of NOS in endothelium‐intact rings increased the % maximal vasoconstriction (% max VC) in MU rats (3.0±0.2 fold). After NOS inhibition, increase in % max VC was attenuated in MS rats (1.5±0.2 fold, p&lt;0.05 vs. MU). Denuded rings showed similar % max VC in both groups. NO basal tone was reduced in MS rats (44.9±7.4 vs. 20.0±4.0, p&lt;0.05). Inhibition of ROS with PEG‐SOD (1000 mU/L) or apocynin (300 μM) decreased the % max VC in MU rats (27.8±3.4 vs. 7.2±0.8 and 9.1±1.9, respectively, p&lt;0.05). Either PEG‐SOD or apocynin did not modify the % max VC in MS rats. These data indicate that MS induces a dysfunction in Ang II mediated signaling in the endothelium, suggesting that early life stress mediates a pro‐contractile vascular phenotype.

Pharmacological characterization of the serotonin receptor mediating contraction in the rat jugular vein

The purpose of this study was to characterize pharmacologically the serotonin (5‐hydroxytryptamine; 5‐HT) receptors mediating contraction in the rat jugular vein (RJV). We hypothesized that 5‐HT2A receptor is not the only receptor mediating contraction in RJV. Endothelium‐intact rings of RJV were placed in a tissue bath for measurement of isometric contractile force induced by cumulative addition of agonists (1nM to 10 μM). The contractions induced by the 5‐HT1A/1B and 5‐HT1A receptor agonists, RU 24969 (pD2=‐log EC50 [M]=6.4±0.05) and 8‐OH‐DPAT (pD2=5.5±0.07), respectively, were not as robust as that induced by 5‐HT (pD2=6.8±0.17). The 5‐HT1B/1D/1F agonist sumatriptan failed to induce contraction. The 5‐HT2A receptor agonist DOI (pD2=7.78±0.12) was more potent, whereas the 5‐HT2B receptor agonist BW723C86 was less potent (pD2=6.17±0.12) when compared to the contraction induced by 5‐HT. The antagonists with high affinity for the 5‐HT2A and 5‐HT2B receptors, ketanserin (30nM) and LY266097 (10nM), respectively, caused a right‐shift of 5‐HT‐induced contraction. The 5‐HT1B/1D receptor antagonist GR127935 (10nM) had no effect on 5‐HT‐induced contraction. We conclude that 5‐HT‐induced contraction of RJV is mediated primarily by 5‐HT2A receptor. The involvement of 5‐HT2B and 5‐HT1A receptor subtypes can not be ruled out and deserve further investigation. By identifying receptor mechanisms, we can better understand how the newly discovered serotonergic system in veins may locally modify contraction.

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

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

Differential involvement of COX1 and COX2 in the vasculopathy associated with the α-galactosidase A-knockout mouse

The lysosomal storage disorder Fabry disease is characterized by excessive globotriaosylceramide (Gb3) accumulation in major organs such as the heart and kidney. Defective lysosomal alpha-galactosidase A (Gla) is responsible for excessive Gb3 accumulation, and one cell sensitive to the effects of Gb3 accumulation is vascular endothelium. Endothelial dysfunction is associated with Fabry disease and excessive cellular Gb3. We previously demonstrated that excessive vascular Gb3 in a mouse model of Fabry disease, the Gla-knockout (Gla(-/0)) mouse, results in abnormal vascular function, which includes abnormal endothelium-dependent contractions, a vascular phenomenon known to involve cyclooxygenase (COX). Therefore, we hypothesized that the vasculopathy in the Gla knockout mouse may be due to a vasoactive COX-derived product. To test this hypothesis, vascular reactivity experiments were performed in aortic rings from wild-type (Gla(+/0)) and Gla(-/0) mice in the presence and absence of specific and nonspecific COX inhibitors. Specific inhibition of COX1 or COX2 in endothelium-intact rings from Gla(-/0) mice decreased overall phenylephrine contractility compared with untreated Gla(-/0) rings, whereas COX inhibitors had no effect on contractility in endothelium-denuded rings. Nonspecific inhibition of COX with indomethacin (10 micromol/l) or COX1 inhibition with valeryl salicylate (3 mmol/l) improved endothelial function in rings from Gla(-/0) mice, but COX2 inhibition with NS-398 (1 micromol/l) further increased endothelial dysfunction in rings from Gla(-/0) mice. These results suggest that, in the Gla(-/0) mice, COX1 and COX2 activity are increased and localized in the endothelium, producing vasopressor and vasorelaxant products, which contribute to the Fabry-related vasculopathy.

Prolonged Cold Storage Diminishes the 5-Hydroxytryptamine- and Potassium Chloride-Mediated Contractions of Rat Thoracic Aorta

Hypothermic preservation of the organ for transplantation causes vascular damage; therefore, the preservation of vascular function is important for the organs to function correctly after transplantation. The aim of the present study is to evaluate the influence of prolonged cold storage (72 hours) on vascular responses to 5-hydroxytryptamine (5-HT) and potassium chloride (KCl), each of which causes receptor-dependent and receptor-independent contractions, respectively. We also examined the protective roles of superoxide dismutase (SOD), L-arginine, the precursor of nitric oxide, iloprost, a synthetic analogue of prostaglandin I(2) with vasodilator functions, or endothelium removal for vascular responses. Endothelium-intact rings were prepared from the rat thoracic aorta, and stored at 4 degrees C for up to 72 hours in Krebs solution alone or Krebs solution that contains SOD, L-arginine or iloprost. The vascular responses were investigated daily. The Analysis of Variance (ANOVA) followed by Dunn test was used for statistical analysis. Being kept in cold in Krebs solution diminished the vascular responses to 5-HT and KCl. The presence of SOD in Krebs solution successfully prevented the decline in these responses, while iloprost or L-arginine partially restored them. In the endothelium-denuded rings, the 5-HT-induced contraction remained protected after 72 hours, whereas the KCl-induced contraction was partially restored. These results indicate that cold preservation declines the 5-HT and KCl-induced vascular responses, which can be partially prevented by iloprost or L-arginine, and can be restored by endothelium removal or SOD. Therefore, superoxide anion and endothelium-derived factors contribute to the decline in the contracting function of the aorta during prolonged cold storage.

Role of Na+-K+ATPase enzyme in vascular response of goat ruminal artery

Objective:To study the role of Na+, K+- ATPase enzyme in the vascular response of goat ruminal artery.Materials and Methods:Ruminal artery was obtained in chilled aerated modified Krebs-Henseleit solution (KHS) from a local slaughterhouse and transported in ice for further processing. The endothelium intact arterial ring was mounted in a thermostatically controlled (37 ± 0.5°C) organ bath containing 20 ml of modified KHS (pH 7.4) bubbled with oxygen (95%) and CO2 (5%) under 2g tension. An equilibration of 90 min was allowed before addition of drugs into the bath. The responses were recorded isometrically in an automatic organ bath connected to PowerLab data acquisition system. In order to examine intact functional endothelium, ACh (10 μM) was added on the 5-HT (1.0 μM) - induced sustained contractile response. Similarly, functional characterization of Na+, K+-ATPase activity was done by K+-induced relaxation (10 μM-10 mM) in the absence and presence of ouabain (0.1 μM/ 0.1 mM), digoxin (0.1 μM) and barium (30 μM).Results:ACh (10−5 M) did not produce any relaxing effect on 5-HT-induced sustained contractile response suggesting that vascular endothelium has no significant influence on the activation of sodium pump by extracellular K+ in ruminal artery. Low concentration of Ba2+ (30 μM) (IC50: 0.479 mM) inhibited K+-induced relaxation suggesting Kir (inward rectifier) channel in part had role in K+-induced vasodilatation in ruminal artery. Vasorelaxant effect of KCl (10 μM-10 mM) in K+-free medium is also blocked by ouabain (0.1 μM and 0.1 mM) (IC50:0.398 mM and IC35: 1.36 mM), but not by digoxin (0.1 μM) (IC50 0.234 mM) suggesting that ouabain sensitive Na+, K+-ATPase isoform is present in the ruminal artery.Conclusion:In the goat ruminal artery functional regulation of sodium pump is partly mediated by K+ channel and ouabain sensitive Na+, K+ ATPase.