Phenylephrine-precontracted Aortic Rings Research Articles

In vitro and ex vivo antioxidant and vasodilatory properties of Syzygium cumini, Piper sarmentosum roxB and Psidium guajava plant extracts from Reunion Island

Ischemic heart disease is the leading cause of cardiovascular death. It may originate from large vessel obstruction or from coronary microvascular dysfunction (CMD). CMD is associated with oxidative stress, which induces endothelial dysfunction and therefore alters vasodilation. The potentially beneficial cardiovascular effects of plant extracts have long been suggested by traditional medicine. We investigated the antioxidant and vasodilatory potential of 5 extracts from 3 plants from Reunion Island on in vitro and ex vivo experimental models. Aqueous extracts (AE) of Piper sarmentosum roxB (PSR) roots, PSR leaves, Psidium guajava (PG) leaves, a methanolic extract (ME) of Syzygium cumini (SC) seeds, and a hydroalcoholic extract (HE) of SC fruits were obtained. Their antioxidant capacities were assessed using DPPH (2,2DiPhenyle-1-Picryl-Hydrazyle). Concentrations of extracts ranged from 5 to 40 μg/mL in order to determine EC50 and T-EC50 values. To assess the vasodilatory potential of the extracts, phenylephrine-precontracted aortic rings from male Sprague Dawley rats with > 60% endothelium-dependent vasodilation were incubated with the extracts (50 μg/mL). Extracts displaying a vasodilatory effect were then used at 0.5, 5, and 50 μg/mL on precontracted aortic rings with (> 60%) or without (< 10%) endothelium. Finally, the pathway involved in endothelium-dependent relaxation by the extract was investigated by preincubation with the nitric oxide synthase (NOS) inhibitor L-NAME. The results are presenting in Table 1 . A high antioxidant capacity was observed for SC seed ME (EC50 = 8.0 μg/mL; T-EC50 = 10 min) and PG AE (EC50 = 9.7 μg/mL; T-EC50 = 15 min). SC seed ME and PG AE also demonstrated a vasodilatory effect (respectively: 64 ± 12% and 55 ± 17% vs. 4 ± 9% for control, P < 0.0001). No vasodilation was observed following endothelium removal. L-NAME significantly inhibited the vasodilatory effect of SC seed ME (32 ± 19% vs. 67 ± 15%, P = 0.003), but not that of PG (38 ± 19% vs. 66 ± 30%, P = 0.7). SC seed and PG demonstrated promising potential to prevent CMD due to their antioxidant properties and endothelium-dependent vasodilatory effects, with the effect of SC seed effect involving endothelial NOS. Further studies are warranted to determine the in vivo potential of these extracts.

Effects of Acrylamide-Induced Vasorelaxation and Neuromuscular Blockage: A Rodent Study.

Acrylamide (ACR), which is formed during the Maillard reaction, is used in various industrial processes. ACR accumulation in humans and laboratory animals results in genotoxicity, carcinogenicity, neurotoxicity, and reproductive toxicity. In this study, we investigated the mechanisms by which ACR may induce vasorelaxation and neuromuscular toxicity. Vasorelaxation was studied using an isolated rat aortic ring model. The aortic rings were divided into the following groups: with or without endothelium, with nitric oxide synthase (NOS) inhibition, with acetylcholine receptor inhibition, and with extracellular calcium inhibition. Changes in tension were used to indicate vasorelaxation. Neuromuscular toxicity was assessed using a phrenic nerve–diaphragm model. Changes in muscle contraction stimulated by the phrenic nerve were used to indicate neuromuscular toxicity. ACR induced the vasorelaxation of phenylephrine-precontracted aortic rings, which could be significantly attenuated by NOS inhibitors. The results of the phrenic nerve–diaphragm experiments revealed that ACR reduced muscle stimulation and contraction through nicotinic acetylcholine receptor (AChR). ACR-induced vasotoxicity was regulated by NOS through the aortic endothelium. Nicotinic AChR regulated ACR-induced neuromuscular blockage.

Role of pregnancy on insulin-induced vasorelaxation: the influence of angiotensin II receptors.

Insulin resistance is a feature of pregnancy and is associated with increased levels of angiotensin II (Ang II) and insulin. Therefore, pregnancy may change insulin-induced vasodilation through changes in Ang II receptors. Insulin-induced vasorelaxation was evaluated in phenylephrine-precontracted aortic rings of pregnant and non-pregnant rats, using a conventional isolated organ preparation. Experiments were performed in thoracic or abdominal aorta rings with or without endothelium in the presence and absence of NG-nitro-L-arginine methyl ester (L-NAME) (10-5 M), losartan (10-7 M), or PD123319 (10-7 M). AT1 and AT2 receptor expressions were detected by immunohistochemistry. Insulin-induced vasodilation was endothelium- and nitric oxide-dependent and decreased in the thoracic aorta but increased in the abdominal segment of pregnant rats. The insulin's vasorelaxant effect was increased by losartan mainly on the thoracic aorta. PD123319 decreased insulin-induced vasorelaxation mainly in the pregnant rat abdominal aorta. AT1 receptor expression was decreased while AT2 receptor expression was increased by pregnancy. In conclusion, pregnancy changes insulin-induced vasorelaxation. Moreover, insulin vasodilation is tonically inhibited by AT1 receptors, while AT2 receptors appear to have an insulin-sensitizing effect. The role of pregnancy and Ang II receptors differ depending on the aorta segment. These results shed light on the role of pregnancy and Ang II receptors on the regulation of insulin-mediated vasodilation.

The vasodilatory effect of the antidiabetic drug linagliptin via inhibition of Rho-associated protein kinase in aortic smooth muscle

AimsThe vasodilatory effects of the anti-diabetic drug, linagliptin in phenylephrine-precontracted aortic rings were investigated. Materials and methodsMale New Zealand White rabbits were used in the experiment and its arterial tone was measured by using myogragh system. Key findingsLinagliptin induced vasodilation in a concentration-dependent manner. The vasodilatory effect of linagliptin was not affected by the absence of the endothelium, or by pretreatment with a nitric oxide synthase inhibitor (L-NAME) or a small-conductance Ca2+-activated K+ channel inhibitor (apamin). Moreover, application of the adenylyl cyclase inhibitor SQ22536, protein kinase A (PKA) inhibitor KT5720, guanylyl cyclase inhibitor ODQ, or protein kinase G (PKG) inhibitor KT5823 did not alter the vasodilatory effect of linagliptin. However, inhibition of Rho-associated protein kinase by Y-27632 significantly attenuated linagliptin-induced vasodilation. Ion channel involvement in the vasodilatory effect of linagliptin was also investigated. Pretreatment with the vascular K+ channel inhibitors glibenclamide (ATP-sensitive K+ channels), Ba2+ (inwardly rectifying K+ channels), 4-AP (voltage-dependent K+ channels), and paxilline (large conductance Ca2+-activated K+ channels) did not affect linagliptin-induced vasodilation. Furthermore, the L-type Ca2+ channel inhibitor, nifedipine, and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor, thapsigargin, did not change the vasodilatory effect of linagliptin. Significance: We suggests that linagliptin-induced vasodilation was mediated by the inhibition of Rho-associated kinase, but not with the endothelium, cAMP-PKA or cGMP-PKG-dependent signaling pathways, K+ channels, Ca2+ influx, or SERCA pump.

Pharmacological characterization of mechanisms involved in the vasorelaxation produced by rosuvastatin in aortic rings from rats with a cafeteria-style diet.

The present study aimed to investigate the possible influence of several inhibitors and blockers on the vascular effect produced by the acute in vitro application of rosuvastatin to phenylephrine-precontracted aortic rings from rats with a semi-solid, cafeteria-style (CAF) diet. It also aimed to examine the effects of rosuvastatin on the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase in aortic rings from rats with a CAF diet. From comparisons of the effect on phenylephrine-precontracted aortic rings extracted from rats with two different diets (a standard and a CAF diet), it was found that 10−9–10−5-mol/L rosuvastatin produced lower concentration-dependent vasorelaxation on rings from the CAF diet group. The vasorelaxant effect was unaffected by the vehicle, but it was significantly attenuated by 10−5-mol/L NG-nitro-l-arginine methyl ester, 10−2-mol/L tetraethylammonium, 10−3-mol/L 4-aminopyridine, 10−7-mol/L apamin plus 10−7-mol/L charybdotoxin, 10−5-mol/L indomethacin, or 10−5-mol/L cycloheximide. Moreover, in aortic rings from rats with a CAF diet, rosuvastatin enhanced the expression of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase. The acute in vitro application of rosuvastatin to phenylephrine-precontracted aortic rings from rats with a CAF diet had a vasorelaxant effect. Overall, the present results suggest that the stimulation of eNOS, the opening of Ca2+-activated and voltage-activated K+ channels, the stimulation of prostaglandin synthesis and enhanced protein levels of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase are involved in this relaxant effect.

Vasorelaxant action of an ethylacetate fraction of Euphorbia humifusa involves NO-cGMP pathway and potassium channels

Ethnopharmacological relevanceEuphorbia humifusa Willd. (EH) is an important traditional Chinese medicine that has commonly been used for treating bacillary dysentery and enteritis in many Asian countries for thousands of years. EH has a wide variety of pharmacological actions such as antioxidant, hypotensive, and hypolipidemic effects. However, the mechanisms involved are to be defined. Aim of the studyThe present study was performed to evaluate the cardiovascular effects of EH in rats. Materials and methodsMethanol extract of EH (MEH) and ethylacetate fraction of the MEH (EEH) was examined for their vascular relaxant effects in phenylephrine-precontracted aortic rings. Effects of EEH on systolic blood pressure and heart rate were tested in Sprague–Dawley rats. ResultsMEH and EEH induced vasorelaxation in a concentration-dependent manner. Endothelium-denudation abolished the EEH-induced vasorelaxation. Pretreatment of the endothelium-intact aortic rings with NG-nitro-l-arginine methylester (l-NAME) and 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ) significantly inhibited the EEH-induced vasorelaxation. EEH increased cGMP levels of the aortic rings in a concentration-dependent manner and the effect was blocked by l-NAME or ODQ. Extracellular Ca2+ depletion and treatments with thapsigargin, Gd3+, and 2-aminoethyl diphenylborinate significantly attenuated the EEH-induced vasorelaxation. Wortmannin markedly attenuated the EEH-induced vasorelaxation. In addition, tetraethylammonium, iberiotoxin, and charybdotoxin, but not apamin, attenuated the EEH-induced vasorelaxation. Glibenclamide, indomethacin, atropine, and propranolol had no effects on the EEH-induced vasorelaxation. Furthermore, EEH decreased systolic blood pressure and heart rate in a concentration-dependent manner in rats. ConclusionsThe present study demonstrates that EEH induces endothelium-dependent vasorelaxation via eNOS-NO-cGMP signaling through the modification of intracellular Ca2+, Ca2+ entry, and large- and intermediate-conductance KCa channel homeostasis. The data also suggest that the Akt-eNOS pathway is involved in the EEH-induced vasorelaxation. EEH induces hypotension and bradycardia in vivo.

Vasorelaxant effects on rat aortic artery by two types of indole alkaloids, naucline and cadamine

Two indole alkaloids, naucline from Nauclea officinalis and cadamine from Ochreinauclea maingayii, were individually evaluated for vasorelaxant effects on phenylephrine-precontracted aortic rings. Naucline induced concentration-dependent relaxation in aortic rings. Respective EC50 values of naucline on aorta rings with/without endothelium did not show a significant difference, indicating that naucline-induced relaxation was independent of the endothelium. In further experiments with various inhibitors, naucline was found to possess inhibitory effects on both voltage-dependent Ca(2+) channel (VDC)- and receptor-operated Ca(2+) channel (ROC)-dependent Ca(2+) influx in smooth muscle. Cadamine showed concentration-dependent relaxation in endothelium-intact aortic rings, which was inhibited by addition of L-NMMA, NOS inhibitor. These results suggested that the vasorelaxant effect of cadamine is partly mediated through the increased release of NO from endothelial cells. In addition to NO involvement, vasorelaxation induced by cadamine was also attributed to inhibition of both VDC- and ROC-dependent Ca(2+) influx.

Vasodilatory effects of ethanol extract of Radix Paeoniae Rubra and its mechanism of action in the rat aorta

Ethnopharmacological relevanceRadix Paeoniae Rubra (RPR) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China. RPR is the radix of either Paeonia lactiflora Pall. or Paeonia veitchii Lynch. RPR has a wide variety of pharmacological actions such as anti-thrombus, anti-coagulation, and anti-atherosclerotic properties, protecting heart and liver. However, the mechanisms involved are to be defined. Aim of the studyThe aim of the present study was to define the effect of Paeonia lactiflora Pall. extracts on vascular tension and responsible mechanisms in rat thoracic aortic rings. Materials and methodsEthanol extract of Paeonia lactiflora Pall. (EPL) was examined for their vascular relaxant effects in isolated phenylephrine-precontracted rat thoracic aorta. ResultsEPL induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Vascular relaxation induced by EPL was significantly inhibited by removal of the endothelium or pretreatment of the rings with NG-nitro-l-arginine methylester (l-NAME) or 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ). Extracellular Ca2+ depletion or diltiazem significantly attenuated EPL-induced vasorelaxation. Modulators of the store-operated Ca2+ entry (SOCE), thapsigargin, 2-aminoethyl diphenylborinate and Gd3+, and an inhibitor of Akt, wortmannin, markedly attenuated the EPL-induced vasorelaxation. Further, the EPL-induced vasorelaxation was significantly attenuated by pretreatment with tetraethylammonium, a non-selective KCa channels blocker, or glibenclamide, an ATP-sensitive K+ channels inhibitor, respectively. Inhibition of cyclooxygenases with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on the EPL-induced vasorelaxation. ConclusionsThe present study suggests that EPL relaxes vascular smooth muscle via endothelium-dependent and Akt- and SOCE-eNOS-cGMP-mediated pathways through activation of both KCa and KATP channels and inhibition of L-type Ca2+ channels.

The mechanism of vasorelaxation induced by ethanol extract of Sophora flavescens in rat aorta

Sophora flavescens (SF) is a known medicinal herb for the treatment of cardiovascular symptoms associated with arrhythmia in China. However, the pharmacological action mechanisms involved have not been well studied. The aim of the present study was to define effects of roots of SF on the vascular tension and responsible mechanisms in rat thoracic aorta. Ethanol extract of the roots of SF (ESF) was examined for their vascular relaxant effect in isolated phenylephrine-precontracted rat thoracic aorta. ESF (0.1-100 μg/ml) induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Endothelium-denudation abolished the ESF-induced vasorelaxation. Pretreatment of the endothelium-intact aortic rings with l-NAME, an inhibitor of nitric oxide synthase, and ODQ, an inhibitor of soluble guanylyl cyclase (sGC), inhibited ESF-induced vasorelaxation. ESF increased cGMP levels of the aortic rings in a concentration-dependent manner and the effect was blocked by l-NAME and ODQ. Inhibition of K(+) channels with glibenclamide and tetraethylammonium, cyclooxygenase inhibition with indomethacin, and β-adrenergic and muscarinic receptors blockade had no effect on the ESF-induced vasorelaxation. These findings suggest that ESF relaxes vascular smooth muscle via endothelium-dependent NO-sGC-cGMP signaling pathway.

Protein kinase A signalling is involved in the relaxant responses to the selective β-oestrogen receptor agonist diarylpropionitrile in rat aortic smooth muscle in vitro

The oestrogen receptor β (ERβ) selective agonist diarylpropionitrile (DPN) relaxes endothelium-denuded rat aorta, but the signalling mechanism is unknown. The aim of this study was to assess whether protein kinase A (PKA) signalling is involved in DPN action. cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry. DPN increased cAMP content and hyperpolarised cell membranes over the same range of concentrations as it relaxed phenylephrine-precontracted aortic rings (10-300 µM). DPN-induced vasorelaxation was largely reduced by the PKA inhibitors Rp-8-Br-cAMPS (8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer) and H-89 (N-(2-bromocynnamyl(amino)ethyl)-5-isoquinoline sulfonamide HCl) (-73%) and by the adenylate cyclase inhibitor MDL12330A (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine)) (-65.5%). Conversely, the PKG inhibitor Rp-8-Br-cGMP was inactive against DPN vasorelaxation. In aortic smooth muscle segments, DPN increased PKA-dependent HSP20 phosphorylation, an effect reversed by H-89. Relaxant responses to DPN were modestly antagonised (-23 to -48% reduction; n=12 per compound) by the potassium channel inhibitors iberiotoxin, PNU-37883A, 4-aminopyridine, or BaCl(2) . All four potassium channel inhibitors together reduced DPN relaxation by 86±9% (n=12) and fully blocked DPN hyperpolarisation. ERβ-dependent relaxation of rat aortic smooth muscle evokes an adenylate cyclase/cAMP/PKA signalling pathway, likely activating the cystic fibrosis transmembrane conductance regulator chloride channel and at least four potassium channels.

Vascular relaxation by ethanol extract of Xanthoceras sorbifolia via Akt- and SOCE-eNOS-cGMP pathways

The aim of the present study was to define the effect of Xanthoceras sorbifolia extracts (XS) on vascular tension and responsible mechanisms in rat thoracic aortic rings. Ethanol extract of the leaves of XS (EXS) was examined for their vascular relaxant effects in isolated phenylephrine-precontracted rat thoracic aorta. EXS (0.1-100 μg/ml) induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Endothelium-denudation abolished EXS-induced vasorelaxation. Pretreatment of the endothelium-intact aortic rings with N(G)-nitro-L-arginine methylester (L-NAME) and 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ) inhibited EXS-induced vasorelaxation. Inhibition of Ca(2+) entry via L-type Ca(2+) channels failed to block the EXS-induced vasorelaxation. Extracellular Ca(2+) depletion significantly attenuated EXS-induced vasorelaxation. Modulators of the store-operated Ca(2+) entry (SOCE), thapsigargin, 2-aminoethyl diphenylborinate (2-APB) and Gd(3+), and an inhibitor of Akt, wortmannin, markedly attenuated the EXS-induced vasorelaxation. EXS increased cGMP levels of the aortic rings in a concentration-dependent manner and the effect was blocked by L-NAME, ODQ, thapsigargin, Gd(3+), 2-APB, and wortmannin. Further, EXS-induced vasorelaxation was significantly attenuated by tetraethylammonium, a non-selective K(ca) channels blocker, but not by glibenclamide, an ATP-sensitive K(+) channels inhibitor. Inhibition of cyclooxygenase with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on EXS-induced vasorelaxation. The present study suggests that EXS relaxes vascular smooth muscle via endothelium-dependent NO-cGMP signaling through activation of the Akt- and SOCE-eNOS-sGC pathways, which may, at least in part, be related to the function of K(+) channels.

AMP‐activated protein kinase (AMPK) mediated relaxation is enhanced in aorta from old versus young Sprague‐Dawley Rats

Activation of AMPK induces vasorelaxation in young, healthy arteries, but it is unknown if this response is intact in arteries from old animals. Here we investigate AMPK‐mediated relaxation in aorta from aged rats. Relaxation dose‐response curves to the AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) were generated in phenylephrine pre‐contracted aortic rings from 12 young (10 wks) and 12 old (85 wks) male Sprague Dawley rats using vascular myography. Area under the dose‐response curves (AUC, arbitrary units) was determined as an index of relaxation. Mechanisms of relaxation were dissected in rings with intact (E+) or mechanically disrupted (E‐) endothelium and with or without pharmacological inhibition of nitric oxide synthase (using N‐nitro‐L‐arginine methyl ester; L‐NAME). Endothelium‐dependent and ‐independent relaxation to AICAR occurred in aortic rings from old (AUC E+: 196±27, E‐: 130±9) and young (AUC E+: 94±5, E‐: 53±4) rats, and responses were enhanced in old versus young rings (P &lt; 0.01 E+, P &lt; 0.01 E‐). In both young and old aorta, the endothelium‐dependent component of relaxation was removed with L‐NAME (AUC OLD: 98±10 P =NS, Young: 52±3 P = NS vs E‐). We demonstrate for the first time that AMPK‐mediated relaxation occurs and is enhanced in aorta from old animals, and that AMPK may be a target to enhance vasomotor function in aged arteries. Funded by NSERC

Water extract of Zanthoxylum piperitum induces vascular relaxation via endothelium-dependent NO-cGMP signaling

Aim of the study The aim of the present study was to define the effects of extracts of leaves of Zanthoxylum piperitum (ZP) on the vascular tension and its mechanisms responsible in rat thoracic aortic rings. Materials and methods Methanol extract of ZP and aqueous fraction of the methanol extract (AZP) were examined for their vascular relaxant effects in isolated phenylephrine-precontracted aortic rings. Results Methanol extract of ZP and aqueous fraction of the methanol extract (AZP) induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Endothelium-denudation abolished the AZP-induced vasorelaxation. Pretreatment of the endothelium-intact aortic rings with N G-nitro- l-arginine methylester (L-NAME) and 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ) inhibited the AZP-induced vasorelaxation. Inhibition of Ca 2+ entry via L-type Ca 2+ channels failed to block the AZP-induced vasorelaxation. Extracellular Ca 2+ depletion slightly but significantly attenuated the AZP-induced vasorelaxation. Thapsigargin significantly attenuated the AZP-induced vasorelaxation. Further, Gd 3+ and 2-aminoethyl diphenylborinate (2-APB), inhibitors of store-operated Ca 2+ entry (SOCE), markedly attenuated the AZP-induced vasorelaxation. Also, wortmannin, an inhibitor of Akt, an upstream signaling molecule of eNOS, attenuated the AZP-induced vasorelaxation. AZP increased cGMP levels of the aortic rings in a concentration-dependent manner and the effect was blocked by L-NAME, ODQ, thapsigargin, Gd 3+, 2-APB, and wortmannin. K + channel inhibition with glibenclamide and tetraethylammonium, cyclooxygenase inhibition with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on the AZP-induced vasorelaxation. Conclusion Taken together, the present study suggests that AZP relaxes vascular smooth muscle via endothelium-dependent activation of NO-cGMP signaling through the Akt- and SOCE-eNOS pathways.

Improvement of impaired endothelial function by tetrahydrobiopterin in stroke-prone spontaneously hypertensive rats

To investigate the role of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, in endothelial function in a model of genetic hypertension, acetylcholine- and sodium nitroprusside (SNP)-induced vasodilator responses were examined in the absence and presence of BH4 in age-matched adult stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats. Acetylcholine-induced depressor responses attenuated significantly in SHRSP compared with those in WKY rats. Acetylcholine-induced relaxations in phenylephrine-precontracted aortic rings of SHRSP were also significantly impaired as compared to those of WKY rats, while SNP-induced relaxations were similar between both strains. In SHRSP, intravenous infusion of BH4 (0.12 mg/kg per min for 20 min following a bolus injection of 0.48 mg/kg) significantly improved vasodilator responses to acetylcholine without affecting those to SNP, but in WKY rats BH4 did not influence those to acetylcholine. BH4 infusion itself had no hemodynamic effect in both strains. However, BH4 levels in plasma and thoracic aorta as well as plasma concentrations of nitrite plus nitrate, metabolites of NO, in SHRSP were all significantly greater than those in WKY rats, suggesting the occurrence of compensatory upregulation of NO synthesis in SHRSP. These results demonstrate that the impaired endothelial function in SHRSP cannot be explained simply by the decrease in absolute amount of BH4.

Isometric contraction increases endothelial nitric oxide synthase activity via a calmodulin antagonist-sensitive pathway in rat aorta

In this work, the possibility that isometric contraction activates endothelial nitric oxide synthase (eNOS) in a calcium/calmodulin (Ca 2+/CaM)-dependent manner was examined in rat thoracic aorta. Step-wise stable contractile responses (precontractions) to phenylephrine were obtained in endothelium-intact and endothelium-denuded aortic rings. The subsequent addition of the NO synthase inhibitor, N Gnitro- l-arginine methyl ester ( l-NAME), or the soluble guanylyl cyclase inhibitor, ODQ, further augmented precontractions in a concentration-dependent manner. The amplitude of l-NAME- and ODQ-induced increases in tone were dependent on the level of precontraction; the maximal increments for l-NAME and ODQ were observed in arteries precontracted with phenylephrine at 67% of its maximal effect. Likewise, in endothelium-intact non-contracted arteries, l-NAME and ODQ induced small but significant increases in tone. Neither l-NAME nor ODQ had any effect in endothelium-denuded preparations. In endothelium-intact aortic rings precontracted with high K + solutions, l-NAME also elicited supplementary contractions dependent on precontraction level. The CaM antagonist, calmidazolium, inhibited in a concentration-dependent, noncompetitive, manner the effects of l-NAME on the tone of endothelium-intact phenylephrine-precontracted aortic rings. These results suggest that isometric contraction increases the activity of eNOS by means of the Ca 2+/CaM complex in rat aorta.

Sexual dimorphism in rabbit aortic endothelial function under acute hyperglycemic conditions and gender-specific responses to acute 17β-estradiol

Epidemiological data suggest that hyperglycemia abrogates the gender-based cardiovascular protection possibly associated with estrogens. This study was designed to investigate 1) whether rabbit aortic rings show gender differences in the development of abnormal endothelium-dependent vasodilation (EDV) under acute hyperglycemic conditions, 2) the potential role of PKC isoforms and superoxide (O2-) in acute hyperglycemia-induced vascular dysfunction, and 3) the effect of acute estrogen administration on hyperglycemia-induced endothelial dysfunction in male and female rabbits. EDV to ACh was determined before and after 3 h of treatment with high glucose (HG) in phenylephrine-precontracted aortic rings from male and female New Zealand White rabbits. Similar experiments were conducted in the presence of inhibitors of PKC-alpha, PKC-beta, and PKC-delta or an O2- scavenger. The effect of acute estrogen administration was evaluated in the presence and absence of HG. Finally, mRNA expression of PKC isoforms was measured by real-time PCR. We found that 1) 3 h of incubation with HG impairs EDV to a greater extent in female than male aorta, 2) inhibition of PKC-beta or O2- prevents HG-induced impairment of EDV in female aorta, 3) acute 17beta-estradiol aggravates HG-induced endothelial dysfunction in female, but not male, aorta, and 4) PKC-alpha and PKC-beta expression are significantly higher in female than male aorta. This study reveals the predisposition of female rabbit aorta to vascular injury under hyperglycemic conditions, possibly via activation of PKC-beta and O2- production. Furthermore, it suggests that, under hyperglycemic conditions, acute estrogen treatment is detrimental to endothelial function in female rabbits.

Effect of ropivacaine on endothelium-dependent phenylephrine-induced contraction in guinea pig aorta

Previous studies have shown that ropivacaine has biphasic vascular effects, causing vasoconstriction at low concentrations and vasorelaxation at high concentrations. This study was designed to examine the role of the endothelium during accidental intravascular absorption of ropivacaine, and to elucidate the mechanisms responsible. Isolated guinea pig aortic rings were suspended for isometric tension recording. The effects of ropivacaine on endothelium-intact and endothelium-denuded aortic rings were assessed. Endothelium-intact aortic rings were pre-contracted with phenylephrine before being exposed to ropivacaine and acetylcholine, in order to generate and compare concentration-response curves. In the absence and presence of yohimbine, propranolol, atropine, indometacin, N(G)-nitro-l-arginine methyl ester (l-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or methylene blue, the contractile response induced by ropivacaine was assessed on endothelium-intact aortic rings pre-contracted with phenylephrine. Ropivacaine (3 x 10(-4) to 10(-2) mol/l) produced vasoconstriction in endothelium-denuded aortic rings, whereas no such response was observed in aortic rings with intact endothelium. In phenylephrine pre-contracted intact aortic rings, ropivacaine induced a greater degree of vasorelaxation than did acetylcholine. Yohimbine, propranolol and atropine all failed to affect the relaxation responses induced by ropivacaine. However, pre-treatment with indometacin (cyclo-oxygenase inhibitor), l-NAME (nitric oxide synthase inhibitor), methylene blue (soluble guanylyl cyclase inhibitor) or ODQ (soluble guanylyl cyclase inhibitor), significantly decreased the ropivacaine-induced relaxation of endothelium-intact aortic rings (3 x 10(-4) to 10(-2) mol/l). Ropivacaine elicits an endothelium-dependent vasorelaxation in phenylephrine pre-contracted aortic rings via the nitric oxide-cyclic guanosine 3',5'-monophosphate pathway and the prostaglandin system.

Abstract 3034: Ramipril Retards Development Of Aortic Valve Stenosis In a Rabbit Model: Role of thioredoxin interacting protein

Background: The prevalence of aortic stenosis (AS) has steadily increased over the past 10 years with little improvement in development of therapeutic interventions. We have previously demonstrated that AS develops within 8 weeks in rabbits treated with vitamin D 2 (vit D 2 25,000IU/4 days weekly), and is associated with biochemical and physiological evidence of induction of endothelial dysfunction. We now conduct an investigation in this model of the effects of the ACE inhibitor ramipril on development of AS, extent of endothelial dysfunction and valvular thioredoxin-interacting protein (Txnip), an important regulator of cellular redox balance. Methods: Male New Zealand White rabbits were treated with vit D 2 alone (n=10) or vit D 2 /ramipril (ramipril about 0.5 mg/kg/day; n=12). Six untreated rabbits served as a normal comparator group. AS development was assessed by echocardiography with measurement of transvalvular velocity ratio (AVv/LVOTv), aortic valve area (AVA), and aortic valve backscatter scores (AV BS ). The extent of endothelial dysfunction, was assessed by plasma asymmetric dimethylarginine (ADMA) concentrations and responsiveness to acetylcholine (ACh) in phenylephrine pre-contracted aortic rings. Positive immunostaining for Txnip was quantified by computer-assisted image analysis. . Results: ( mean ±SEM) Echocardiographic data indicated increased AVv/LVOTv, decreased AVA and increased AV BS in the vit D 2 treated rabbits over 8 weeks .Vit D 2 /ramipril attenuated AVv/LVOTv (p&lt;0.05) and AVA (p&lt;0.05, 2-way ANOVA for both); with borderline decreases in AV BS (17.8 ± 2.1dB vs 11.9 ± 2.4 dB, p=0.08). ADMA concentrations were decreased (p&lt;0.01, 2-way ANOVA) in the Vit D 2 /ramipril group and vascular response to ACh and AV BS were inversely correlated (r 2 = 0.9, p&lt;0.0001). Valve Txnip was diminished for vit D 2 /ramipril (0.44±0.03 % total area,) vs vit D 2 (0.89±0.18 %), p=0.03. Conclusion: In this model of AS, ramipril significantly retarded progression of AS and ameliorated endothelial dysfunction. Furthermore, ramipril reversed vit D 2 -induced increases in valvular Txnip, implying an improvement of intracellular antioxidant status. These data add to the rationale for formal clinical evaluation of ACE inhibitor effects in AS.

Vascular effects of long-term propranolol administration after chronic nitric oxide blockade

Long-term propranolol treatment reduces arterial blood pressure in hypertensive individuals mainly by reducing peripheral vascular resistance, but mechanisms underlying their vasodilatory effect remain poorly investigated. This study aimed to investigate whether long-term propranolol administration ameliorates the impairment of relaxing responses of aorta and mesenteric artery from rats made hypertensive by chronic nitric oxide (NO) deficiency, and underlying mechanisms mediating this phenomenon. Male Wistar rats were treated with N(omega)-Nitro-L-arginine methyl ester (L-NAME; 20 mg/rat/day) for four weeks. DL-Propranolol (30 mg/rat/day) was given concomitantly to L-NAME in the drinking water. Treatment with L-NAME markedly increased blood pressure, an effect largely attenuated by DL-propranolol. In phenylephrine-precontracted aortic rings, the reduction of relaxing responses for acetylcholine (0.001-10 microM) in L-NAME group was not modified by DL-propranolol, whereas in mesenteric rings the impairment of acetylcholine-induced relaxation by L-NAME was significantly attenuated by DL-propranolol. In mesenteric rings precontracted with KCl (80 mM), DL-propranolol failed to attenuate the impairment of acetylcholine-induced relaxation by L-NAME. The contractile responses to extracellular CaCl2 (1-10 mM) were increased in L-NAME group, and co-treatment with DL-propranolol reduced this response in both preparations in most Ca2+ concentrations used. The NO2/NO3 plasma levels and superoxide dismutase (SOD) activity were reduced in L-NAME-treated rats, both of which were significantly prevented by DL-propranolol. In conclusion, propranolol-induced amplification of the relaxation to acetylcholine in mesenteric arteries from L-NAME-treated rats is sensitive to depolarization. Additional mechanisms involving blockade of Ca2+ entry in the vascular smooth muscle and increase in NO bioavailability contributes to beneficial effects of long-term propranolol treatment.

Comparison of the effect of simvastatin, spironolactone and L-arginine on endothelial function of aorta in hereditary hypertriglyceridemic rats

Hereditary hypertriglyceridemic (hHTG) rats are characterized by increased blood pressure and impaired endothelium-dependent relaxation of conduit arteries. The aim of this study was to investigate the effect of long-term (4 weeks) treatment of hHTG rats with three drugs which, according to their mechanism of action, may be able to modify the endothelial function: simvastatin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase), spironolactone (an antagonist of aldosterone receptors) and L-arginine (a precursor of nitric oxide formation). At the end of fourth week the systolic blood pressure in the control hHTG group was 148+/-2 mm Hg and in control normotensive Wistar group 117+/-3 mm Hg. L-arginine failed to reduce blood pressure, but simvastatin (118+/-1 mm Hg) and spironolactone (124+/-4 mm Hg) treatment significantly decreased the systolic blood pressure. In isolated phenylephrine-precontracted aortic rings from hHTG rats endothelium-dependent relaxation was diminished as compared to control Wistar rats. Of the three drugs used, only simvastatin improved acetylcholine-induced relaxation of the aorta. We conclude that both simvastatin and spironolactone reduced blood pressure but only simvastatin significantly improved endothelial dysfunction of aorta. Prominent increase in the expression of eNOS in large conduit arteries may be the pathophysiological mechanism underlying the protective effect of simvastatin in hHTG rats.