Contraction Of Aortic Rings Research Articles

Non-steroidal anti-inflammatory drugs attenuate the vascular responses in aging metabolic syndrome rats.

Metabolic syndrome (MS) and aging are low-grade systemic inflammatory conditions, and inflammation is a key component of endothelial dysfunction. The aim of this study was to investigate the effects of non-steroidal anti-inflammatory drugs (NSAIDs) upon the vascular reactivity in aging MS rats. MS was induced in young male rats by adding 30% sucrose in drinking water over 6, 12, and 18 months. When the treatment was finished, the blood samples were collected, and aortas were dissected out. The expression of COX isoenzymes and PLA2 in the aortas was analyzed using Western blot analysis. The contractile responses of aortic rings to norepinephrine (1 μmol/L) were measured in the presence or absence of different NSAIDs (10 μmol/L for each). Serum levels of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) in control rats were remained stable during the aging process, whereas serum IL-6 in MS rats were significantly increased at 12 and 18 months. The levels of COX isoenzyme and PLA2 in aortas from control rats increased with the aging, whereas those in aortas from MS rats were irregularly increased with the highest levels at 6 months. Pretreatment with acetylsalicylic acid (a COX-1 preferential inhibitor), indomethacin (a non-selective COX inhibitor) or meloxicam (a COX-2 preferential inhibitor) decreased NE-induced contractions of aortic rings from MS rats at all the ages, with meloxicam being the most potent. Acetylsalicylic acid also significantly reduced the maximum responses of ACh-induced vasorelaxation of aortic rings from MS rats, but indomethacin and meloxicam had no effect. NSAIDs can directly affect vascular responses in aging MS rats. Understanding the effects of NSAIDs on blood vessels may improve the treatment of cardiovascular diseases and MS in the elders.

<i>Zostera noltii</i> extract lowers blood glucose and restores vascular function in diabetic rats

The antidiabetic effect of seagrass Zostera noltii extract was investigated through a crosstalk between its antioxidant and vasoprotective properties. The extract was orally administered to alloxan-diabetic rats (50, 150, 250 mg/kg body weight). Serum glucose was determined; liver and kidney functions, body weight, total leukocyte counts were measured; liver oxidative markers were assayed. Acetylcholine, phenylephrine and 5-HT responses were tested. eNOS levels and generation of ROS in aortic tissue were quantified. The extract of Z. noltii lowered blood glucose in all tested dose levels. Extract at a concentration of 50 mg/kg failed to preserve the levels of antioxidants and did not alter lipid peroxidation whereas higher doses improved liver oxidative status. Impaired acetylcholine relaxations, augmented phenylephrine and 5-HT contractions in alloxan-diabetic aortic rings were restored by Z. noltii treatment. This recovery was accompanied by increased eNOS synthesis and a reduction in ROS generation. The extract lowers blood glucose and prevents hyperglycemia-induced endothelial dysfunction in alloxan-diabetic rats.

Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas

Elsholtzia splendens (ES) is, rich in flavonoids, used to repair copper contaminated soil in China, which has been reported to benefit cardiovascular systems as folk medicine. However, few direct evidences have been found to clarify the vasorelaxation effect of total flavonoids of ES (TFES). The vasoactive effect of TFES and its underlying mechanisms in rat thoracic aortas were investigated using the organ bath system. TFES (5–200mg/L) caused a concentration-dependent vasorelaxation in endothelium-intact rings, which was not abolished but significantly reduced by the removal of endothelium. The nitric oxide synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (100μM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (30μM) significantly blocked the endothelium-dependent vasorelaxation of TFES. Meanwhile, NOS activity in endothelium-intact aortas was concentration-dependently elevated by TFES. However, indomethacin (10μM) did not affect TFES-induced vasorelaxation. Endothelium-independent vasorelaxation of TFES was significantly attenuated by KATP channel blocker glibenclamide. The accumulative Ca2+-induced contraction in endothelium-denuded aortic rings primed with KCl or phenylephrine was markedly weakened by TFES. These results revealed that the NOS/NO/cGMP pathway is likely involved in the endothelium-dependent vasorelaxation induced by TFES, while activating KATP channel, inhibiting intracellular Ca2+ release, blocking Ca2+ channels and decreasing Ca2+ influx into vascular smooth muscle cells might contribute to the endothelium-independent vasorelaxation conferred by TFES.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas.

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

FGF23 directly impairs endothelium-dependent vasorelaxation by increasing superoxide levels and reducing nitric oxide bioavailability.

Fibroblast growth factor 23 (FGF23) is secreted primarily by osteocytes and regulates phosphate and vitamin D metabolism. Elevated levels of FGF23 are clinically associated with endothelial dysfunction and arterial stiffness in chronic kidney disease (CKD) patients; however, the direct effects of FGF23 on endothelial function are unknown. We hypothesized that FGF23 directly impairs endothelial vasorelaxation by hindering nitric oxide (NO) bioavailability. We detected expression of all four subtypes of FGF receptors (Fgfr1-4) in male mouse aortas. Exogenous FGF23 (90-9,000 pg/ml) did not induce contraction of aortic rings and did not relax rings precontracted with PGF2α. However, preincubation with FGF23 (9,000 pg/ml) caused a ∼36% inhibition of endothelium-dependent relaxation elicited by acetylcholine (ACh) in precontracted aortic rings, which was prevented by the FGFR antagonist PD166866 (50 nM). Furthermore, in FGF23-pretreated (9,000 pg/ml) aortic rings, we found reductions in NO levels. We also investigated an animal model of CKD (Col4a3(-/-) mice) that displays highly elevated serum FGF23 levels and found they had impaired endothelium-dependent vascular relaxation and reduced nitrate production compared with age-matched wild types. To elucidate a mechanism for the FGF23-induced impairment, we measured superoxide levels in endothelial cells and aortic rings and found that they were increased following FGF23 treatment. Crucially, treatment with the superoxide scavenger tiron reduced superoxide levels and also restored aortic relaxation to ACh. Therefore, our data suggest that FGF23 increases superoxide, inhibits NO bioavailability, and causes endothelial dysfunction in mouse aorta. Together, these data provide evidence that high levels of FGF23 contribute to cardiovascular dysfunction.

Effects of reduced glutathione on contractile and diastolic functions of thoracic aorta of rats with obstructive jaundice

Objective To evaluate the effects of reduced glutathione (GSH) on contractile and diastolic functions of the thoracic aorta of rats with obstructive jaundice.Methods Twenty-four adult male Sprague-Dawley rats,weighing 200-250 g,were randomly divided into 4 groups (n =6 each) using a random number table:sham operation group (group S),GSH group,bile duct ligation group (group BDL),and GSH + bile duct ligation group (group GSH + BDL).In GSH and GSH + BDL groups,GSH 300 mg/kg was infused via stomach everyday for 7 consecutive days,while the equal volume of normal saline was given in the other two groups.The common bile duct was ligated in the animals on 1 d after the end of intragastric administration in GSH and GSH + BDL groups.In GSH and GSH + BDL groups,after the model was established,GSH 300 mg/kg was infused via stomach everyday for 7 consecutive days,while the equal volume of normal saline was given in the other two groups.On 7th day after the model was extablished,blood samples were collected for determination of the levels of serum total bilirubin (TB),alanine aminotransferase (ALT) and aspartate aminotransferase (AST),tumor necrosis factor-alpha (TNF-α),interleukin-lβ (IL-1β) and 3-nitrotyrosine (3-NT),malondialdehyde (MDA),nitric oxide (NO) and GSH.The thoracic aortic rings were obtained and perfused with different concentrations of norepinephrine (NE),acetylcholine (Ach) and sodium nitroprusside (SNP).The maximum contraction and dilatation of aortic rings were recorded and the percentage was calculated.Some thoracic aortic rings were obtained again and perfused with different concentrations of NE after removal of the endothelium or after being perfused with L-NAME (nitricoxide synthase inhibitor).The maximum contraction of aortic rings was recorded and the percentage was calculated.Results Compared with group S,the serum levels of TB,ALT,AST,TNF-α,IL-1β,MDA,3-NT,NO and GSH were significantly increased in group BDL,and the serum levels of TB,ALT,AST,MDA,GSH and NO were increased in group BDL + GSH,the percentage of the maximum contraction amplitude of aortic rings in response to NE was decreased,and the percentage of the maximum dilatation amplitude of aortic rings in response to Ach and SNP were decreased in groups BDL and BDL + GSH,and no significant changes were found in the parameters mentioned above in group GSH.Compared with group BDL,the serum levels of TB,ALT,AST,TNF-α,IL-1β,MDA,3-NT and NO were significantly decreased,the level of GSH was increased,the percentage of the maximum contraction amplitude of aortic rings in response to NE was increased,the percentage of the maximum dilatation amplitude of aortic rings in response to Ach was increased,and no significant change was found in the percentage of the maximum dilatation amplitude of aortic rings in response to SNP in group BDL + GSH.Compared with the aortic rings from which the endothelium was removed or which were perfused with L-NAME in group BDL,no significant change was found in the percentage of the maximum contraction amplitude of aortic rings after removal of the endothelium or after being perfused with L-NAME in response to NE in group BDL + GSH.Conclusion GSH can improve the contractile and diastolic functions of thoracic aorta of rats with obstructive jaundice and this effect depends on the vascular endothelium. Key words: Jaundice, obstructive ; Glutathione ; Aorta, thoracic

Reprint of “Induction of heme oxygenase-1 ameliorates vascular dysfunction in streptozotocin-induced type 2 diabetic rats”

AimsTo explore the effects of heme oxygenase-1 (HO-1) on vascular dysfunction in high fat diet streptozotocin-induced type 2 diabetic (T2D) rats. MethodsRats received a high-fat diet followed by a low dose of streptozotocin (30mg/kg) to induce T2D. T2D rats were treated with hemin (1, 5, or 25mg/kg) or carbon monoxide-releasing molecule-2 (CORM-2, 5mg/kg) for 4weeks. Isometric contractions of aortic rings were measured. The expression of cyclooxygenase-2 (COX-2) and activities of HO, SOD, and MDA were evaluated. ResultsThe fasting blood glucose, blood insulin levels, and IR index in T2D rats were higher than those in the control group, which were ameliorated by HO-1 inducer hemin. The antidiabetic effect was accompanied by enhanced HO activity. The vascular relaxation response to ACh was decreased in T2D rats, while treatment with hemin could prevent such decrease in vasorelaxation. An increase in COX-2 expression was found in the aortas of T2D rats. Treatment of T2D rats with COX-2 inhibitor NS398 restored ACh-induced vasodilation. COX-2 overexpression in T2D rats was inhibited by hemin. Hemin treatment also inhibited the decline of SOD activity and the increase of MDA content in the aorta of T2D rats. CORM-2, an agent which releases the HO-1 product CO, could mimic the beneficial effect of hemin. ConclusionInduction of HO-1 with hemin ameliorates the abnormality of endothelium-dependent vascular relaxation in T2D rats. A possible mechanism involves suppression of reactive oxygen species production and inhibition of COX-2 up-regulation induced by diabetes mellitus.

S7-2 H2S induced decomposition of nitroso-compounds and consequent biological effect

H2S signaling pathways interact with NO signaling pathways. To contribute to the understanding of the cross-talk between these pathways, we studied H2S induced NO release from nitroso-compounds in vitro and the biological effects of the reaction products. It was observed that H2S released NO from brain homogenate or cultured cells, indicating that biological systems contain nitroso-compounds from which H2S can induce NO release. In an in vitro model, the H2S donor Na2S decomposes S-nitrosoglutation (GSNO), which results in the formation of polysulfides S n 2 - and (SSNO−). Bolus application of a S n 2 - and H2S mixture at microM concentrations had significantly pronounced effect in vivo on rat hemodynamic parameters compared to H2S alone. In ex-vivo models, SSNO− relaxed phenylephrine induced aortic and mesenteric ring contraction. Its effect was faster and more pronounced in comparison to GSNO. As detected by spin trap EPR technique, SSNO− released NO faster than GSNO and scavenged the cPTIO radical. S n 2 - and SSNO− both modulated the activities of calcium RyR2 and chloride channels. In conclusion, the exogenously produced products of H2S-GSNO interaction, S n 2 - and SSNO−, had biological effects in vivo, ex-vivo and in vitro. However, detection of possible endogenous products of H2S-GSNO interactions is a challenge for future research. This work was supported by APVV-0074-11 and VEGA 2/0050/13.

Abstract 514: Knockout of Rictor in Adipocytes Compromises Anticontractile Function of PVAT and Increases Mean Arterial Pressure

Introduction: Mammalian target of rapamycin (mTOR) is a kinase found in two distinct complexes: mTORC1 and mTORC2. The latter is activated by growth factors such as insulin and characterized by the adaptor protein rictor. mTORC2 controls essential functions in white adipose tissue, while little is known about its role in perivascular adipose tissue (PVAT), surrounding most arteries. PVAT is an important regulator of the vascular tone and we have shown that mTORC2 is necessary for normal PVAT function. In the present study we assessed whether adipose mTORC2 contributes to blood pressure regulation and investigated the diurnal expression of mTORC2 phosphorylation targets and circadian clock in PVAT. Methods: Experiments were performed with male adipose-specific rictor knockout mice ( rictor ad-/- ) and control littermates (18-23 weeks of age). Vascular function was assessed ex vivo using aortic rings with or without PVAT treated with 5-hydroxytrypthamine (5-HT; 10 -9 - 3x10 -6 mol/l). Blood pressure recordings were performed using radiotelemetry monitoring hemodynamic parameters over 7 days (n = 5-6). mRNA and protein levels were analyzed by performing qRT-PCR and Western Blot. Results: In rictor ad-/- mice, vascular maximal contractions of aortic rings in the presence of PVAT were 2-fold higher after stimulation with 5-HT compared with control mice (43.4% ± 6.8% vs. 26.0% ± 3.5%, n= 13 ) . Removal of PVAT resulted in increased contraction with similar maximum levels in both mice groups. Blood pressure recordings revealed a significant increase in the 24-h average of mean arterial pressure (103.9 ± 1.1 vs. 98.5 ± 1.4 mmHg) and diastolic arterial pressure (94.6 ± 1.4 vs. 90.2 ± 0.6 mmHg) in rictor ad -/- mice, while systolic arterial pressure and pulse pressure were not significantly different between groups. Interestingly, differences in mean and diastolic arterial pressure were slightly higher during dark as compared to light period. Expression levels of clock genes were similar while expression of mTORC2 phosphorylation targets such as Foxo1 and Sgk1 was dampened in rictor ad-/- mice. Conclusion: mTORC2 in adipose tissue regulates mean arterial pressure potentially by affecting diurnal expression and activation of phosphorylation targets such as Foxo1.

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K+

It has been reported that bitter tastants decrease blood pressure and relax precontracted vascular smooth muscle. However, the underlying mechanisms remain unclear. The aim of the present study was to determine the mechanism underlying the vasorelaxant effect of the bitter tastants. Thoracic aortic rings were isolated from Wistar rats and contractions were measured using an isometric myograph. Intracellular Ca(2+) ([Ca(2+)]i) in single rat thoracic aortic smooth muscle cells was recorded by calcium imaging. Calcium currents in single cells were recorded using patch-clamp techniques. High K(+) (140 mmol/L) induced contractions in rat thoracic aortic rings that were inhibited by 3 mmol/L chloroquine, 3 mmol/L denatonium and 10 μmol/L nifedipine. In single rat thoracic aortic smooth muscle cells, high K(+) increased [Ca(2+)]i and this effect was also blocked by 3 mmol/L chloroquine and 10 μmol/L nifedipine. Under Ca(2+) -free conditions, high K(+) failed to induce contractions in rat thoracic aortic rings. On its own, chloroquine had no effect on the muscle tension of rat aortic rings and [Ca(2+) ]i. The vasorelaxant effects of chloroquine on precontracted rat thoracic aortic rings were not altered by either 1 μg/mL pertussis toxin (PTX), an inhibitor of Gαo/i-protein, or 1 mmol/L gallein, an inhibitor of Gβγ-protein. The results of patch-clamp analysis in single cells indicate that 1 mmol/L chloroquine blocks voltage-dependent L-type Ca(2+) channel (VDLCC) currents from both extracellular and intracellular sides. Together, the results indicate that chloroquine can block VDLCC, independent of PTX- and gallein-sensitive G-proteins, resulting in relaxation of high K(+)-precontracted thoracic aortic smooth muscle.

PT339 The Ang 1-7 mediated vascular and coronary reactivity in diabetic endothelial dysfunction

Introduction: Ang 1-7 appeares as an intrigant peptide regulating AT1 receptor activity and effects. Objectives: The in vitro evaluation of rat aorta and coronary system response on action of the different neuroendocrine factors, inclusively Ang 1-7, in diabetes induced endothelial dysfunction. Methods: Diabetes induced endothelial dysfunction (DED) was reproduced by streptozotocin administration in rats. Using the models of isolated aorta ring and isovolumic heart perfusion the vascular response (relaxant or constricting) and coronary flux were determined on action of acetylcholine (Ach), bradykinin, Ang 1-7, Ang II (inclusively in condition of mas receptor blockade by A779), hydrogen peroxide, norepinephrine and endothelin-1. Results: The Ach action up on phenylephrine induced aorta ring contraction manifested in DED by significant vasorelaxation reduction by up to 36% vs control pattern. Remarkably, Ang 1-7 (10-10 M) has induced an aortic ring relaxation similarly to control level, achieving a range of 11,4-26,5%. The Ang II induced constrictor plateau is significantly elevated in DED by 51,9%. However, it decreases by 32,8% when a premedication by Ang 1-7 is performed, and, contrarily, raises by 36,4% when mas receptors are blocked by A779. Norepinephrine and endothelin-1 have induced in DED an aorta contraction increasing by 34-42%. The analogous events were fixed in heart. Thus, functional coronary reserve is reduced in DED on Ach and bradykinin action by 38 and 31%, respectively. Nevertheless, coronary reserve is not affected during Ang 1-7 action compared to control index: 8,84 vs 9,86% (p>0,05). Coronary flux on Ang II action is decreased more pronounced by 46%, but the decline is limited by almost 50% in condition of Ang 1-7 administration. The mas receptor blocking led to Ang II induced coronary flow diminution more by 52%. Interestingly, the hyperpolarization induced coronary dilation level related to hydrogen peroxide action is not impaired in DED. Conclusion: 1. Vasorelaxation and coronary dilation effects of Ang 1-7 are not impaired in diabetes induced endothelial dysfunction, event that may be emphasized as compensator pattern considering the fact of affected cholinergic vascular response. 2. Ang 1-7 significantly reduces Ang II induced vasoand coronary constricting effects, but mas receptor blocking, contrarily, leads to their augmentation. Disclosure of Interest: None Declared

PT340 Age-related impairment in androgen stimulation of vascular endothelial growth factor-mediated angiogenesis

Introduction: Ang 1-7 appeares as an intrigant peptide regulating AT1 receptor activity and effects. Objectives: The in vitro evaluation of rat aorta and coronary system response on action of the different neuroendocrine factors, inclusively Ang 1-7, in diabetes induced endothelial dysfunction. Methods: Diabetes induced endothelial dysfunction (DED) was reproduced by streptozotocin administration in rats. Using the models of isolated aorta ring and isovolumic heart perfusion the vascular response (relaxant or constricting) and coronary flux were determined on action of acetylcholine (Ach), bradykinin, Ang 1-7, Ang II (inclusively in condition of mas receptor blockade by A779), hydrogen peroxide, norepinephrine and endothelin-1. Results: The Ach action up on phenylephrine induced aorta ring contraction manifested in DED by significant vasorelaxation reduction by up to 36% vs control pattern. Remarkably, Ang 1-7 (10-10 M) has induced an aortic ring relaxation similarly to control level, achieving a range of 11,4-26,5%. The Ang II induced constrictor plateau is significantly elevated in DED by 51,9%. However, it decreases by 32,8% when a premedication by Ang 1-7 is performed, and, contrarily, raises by 36,4% when mas receptors are blocked by A779. Norepinephrine and endothelin-1 have induced in DED an aorta contraction increasing by 34-42%. The analogous events were fixed in heart. Thus, functional coronary reserve is reduced in DED on Ach and bradykinin action by 38 and 31%, respectively. Nevertheless, coronary reserve is not affected during Ang 1-7 action compared to control index: 8,84 vs 9,86% (p>0,05). Coronary flux on Ang II action is decreased more pronounced by 46%, but the decline is limited by almost 50% in condition of Ang 1-7 administration. The mas receptor blocking led to Ang II induced coronary flow diminution more by 52%. Interestingly, the hyperpolarization induced coronary dilation level related to hydrogen peroxide action is not impaired in DED. Conclusion: 1. Vasorelaxation and coronary dilation effects of Ang 1-7 are not impaired in diabetes induced endothelial dysfunction, event that may be emphasized as compensator pattern considering the fact of affected cholinergic vascular response. 2. Ang 1-7 significantly reduces Ang II induced vasoand coronary constricting effects, but mas receptor blocking, contrarily, leads to their augmentation. Disclosure of Interest: None Declared

Cannabinoid receptor type 1 activation by arachidonylcyclopropylamide in rat aortic rings causes vasorelaxation involving calcium-activated potassium channel subunit alpha-1 and calcium channel, voltage-dependent, L type, alpha 1C subunit

Cannabinoids are key regulators of vascular tone, some of the mechanisms involved include the activation of cannabinoid receptor types 1 and 2 (CB); the transient receptor potential cation channel, subfamily V, member 1 (TRPV1); and non-(CB1)/non-CB2 receptors. Here, we used the potent, selective CB1 agonist arachidonylcyclopropylamide (ACPA) to elucidate the mechanism underlying vascular tone regulation. Immunohistochemistry and confocal microscopy revealed that CB1 was expressed in smooth muscle and endothelial cells in rat aorta. We performed isometric tension recordings in aortic rings that had been pre-contracted with phenylephrine. In these conditions, ACPA caused vasorelaxation in an endothelium-independent manner. To confirm that the effect of ACPA was mediated by CB1 receptor, we repeated the experiment after blocking these receptors with a selective antagonist, AM281. In these conditions, ACPA did not cause vasorelaxation. We explored the role of K+ channels in the effect of ACPA by applying high-K+ solution to induce contraction in aortic rings. In these conditions, the ACPA-induced vasorelaxation was about half that observed with phenylephrine-induced contraction. Thus, K+ channels were involved in the ACPA effect. Furthermore, the vasorelaxation effect was similarly reduced when we specifically blocked calcium-activated potassium channel subunit alpha-1 (KCa1.1) (MaxiK; BKCa) prior to adding ACPA. Finally, ACPA-induced vasorelaxation was also diminished when we specifically blocked the calcium channel, voltage-dependent, L type, alpha 1C subunit (Cav1.2). These results showed that ACPA activation of CB1 in smooth muscle caused vasorelaxation of aortic rings through a mechanism involving the activation of KCa1.1 and the inhibition of Cav1.2.

Induction of heme oxygenase-1 ameliorates vascular dysfunction in streptozotocin-induced type 2 diabetic rats

AimsTo explore the effects of heme oxygenase-1 (HO-1) on vascular dysfunction in high fat diet streptozotocin-induced type 2 diabetic (T2D) rats. MethodsRats received a high-fat diet followed by a low dose of streptozotocin (30mg/kg) to induce T2D. T2D rats were treated with hemin (1, 5, or 25mg/kg) or carbon monoxide-releasing molecule-2 (CORM-2, 5mg/kg) for 4weeks. Isometric contractions of aortic rings were measured. The expression of cyclooxygenase-2 (COX-2) and activities of HO, SOD, and MDA were evaluated. ResultsThe fasting blood glucose, blood insulin levels, and IR index in T2D rats were higher than those in the control group, which were ameliorated by HO-1 inducer hemin. The antidiabetic effect was accompanied by enhanced HO activity. The vascular relaxation response to ACh was decreased in T2D rats, while treatment with hemin could prevent such decrease in vasorelaxation. An increase in COX-2 expression was found in the aortas of T2D rats. Treatment of T2D rats with COX-2 inhibitor NS398 restored ACh-induced vasodilation. COX-2 overexpression in T2D rats was inhibited by hemin. Hemin treatment also inhibited the decline of SOD activity and the increase of MDA content in the aorta of T2D rats. CORM-2, an agent which releases the HO-1 product CO, could mimic the beneficial effect of hemin. ConclusionInduction of HO-1 with hemin ameliorates the abnormality of endothelium-dependent vascular relaxation in T2D rats. A possible mechanism involves suppression of reactive oxygen species production and inhibition of COX-2 up-regulation induced by diabetes mellitus.

Effects of oxidative stress on endothelial modulation of contractions in aorta from renal hypertensive rats

BackgroundEndothelial dysfunction is linked to exaggerated production of superoxide anions. This study was conducted to examine the effects of oxidative stress on endothelial modulation of contractions in chronic two-kidney, one-clip (2K1C) renal hypertensive rats. MethodsThe 2K1C hypertension was induced by clipping the left renal artery; age-matched rats receiving sham treatment served as controls. Thoracic aortae were isolated and mounted in tissue baths for measurement of isometric tension. ResultsNorepinephrine-induced contraction was augmented by the removal of the endothelium, which was more pronounced in sham rats than in 2K1C rats. Nω-nitro-L-arginine methyl ester, an inhibitor of nitric oxide production, had a similar augmenting effect. Vitamin C inhibited the contraction in aortic rings with intact endothelium from 2K1C rats but not from sham rats. The contraction was also suppressed by treatment with diphenyleneiodonium or apocynin, inhibitors of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase, in the aortae with intact endothelium from 2K1C rats but not in those from sham rats. Superoxide anions generated by xanthine oxidase/hypoxanthine enhanced the contraction in the aortae with intact endothelium from sham rats, but had no effect in 2K1C rats. Enhanced contractile responses to norepinephrine by xanthine oxidase/hypoxanthine in sham rats were reversed by vitamin C. ConclusionThese results suggest that the effect on endothelial modulation of endothelium-derived nitric oxide is impaired in 2K1C hypertension. The impairment is, at least in part, related to increased production of superoxide anions by NADH/NADPH oxidase.

MC-002 exhibits positive effects against platelets aggregation and endothelial dysfunction through thromboxane A 2 inhibition

Thromboxane A2 (TXA2) induces platelet aggregation and vasoconstriction, and agents that inhibit TXA2 production or interaction with receptors may exert potential application in stroke therapy. To illustrate the platelet aggregation antagonistic and endothelial protective effect of (E) - 3 - (3 - methoxy - 4 - ((3, 5, 6 - trimethylpyrazin - 2 - yl) methoxy) phenyl) sodium acrylate (MC-002) through TXA2 inhibition and underline mechanisms. Platelets aggregation and thoracic aorta ring contraction of rabbits were induced by U46619. Human umbilical vein endothelial cells (HUVECs) were further applied to explore the protective effect of MC-002 on endothelium when exposed to tumor necrosis factor - α (TNF-α). MTT method was used to assess cell damage, and ELISA analysis was exerted to estimate nitrogen monoxide (NO), endothelin-1 (ET-1), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) releasing. Fluorescence spectrophotometry was conducted to determine intracellular calcium concentration ([Ca(2+)]i), and western blotting method was applied to evaluate the protein expressions of intracellular adhesion molecule-1 (ICAM-1), P-selectin and nuclear factor-kappa B (NF-κB). TXA2 analog U46619 mediated obvious platelet aggregation and vasoconstriction. MC-002 inhibited platelet aggregation through administration in vivo and incubation with platelet in vitro, and relaxed aorta ring in endothelium dependent manner. MC-002 alleviated cell damage, [Ca(2+)]i overload, ET-1 overexcretion and TXB2 activation, but improved NO availability reduction in HUVECs treated with TNF-α. Furthermore, MC-002 downregulated ICAM-1, P-selectin and NF-κB overexpression induced by TNF-α. In conclusion, MC-002 exerted antiplatelet aggregation effect through TXA2 inhibition and relieved inflammatory injury of endothelial cells through NF-κB signal pathway.

Molecular interactions of serotonin (5-HT) and endothelin-1 in vascular smooth muscle cells: in vitro and ex vivo analyses.

Elevated levels of serotonin (5-HT) and endothelin-1 (ET-1) may be involved in cardiovascular complications of diabetes mellitus. Data suggest supraphysiological concentrations of 5-HT (10(-6) M) potentiate the ability of ET-1 to stimulate DNA synthesis and vascular smooth muscle cell (VSMC) proliferation in vitro via activation of mitogen-activated protein kinase (p42/44 MAPK) and Janus kinase 2 (JAK2) pathways. Additionally, 5-HT enhances agonist-induced contractions via p42/44 MAPK and an unknown tyrosine kinase. However, the exact mechanisms of the 5-HT/ET-1 interactions and whether these effects occur at physiological levels (10(-9) M) are unknown. Therefore, we hypothesized that interactions between 5-HT and ET-1 at physiological concentrations in VSMC enhanced activation of both p42/44 MAPK and JAK2 pathways contributing to vascular growth and contractile responses. With the use of rat VSMC and Western blot analysis, our data suggest no effect of acute (30 min) preincubation with 5-HT (10(-9) M) and/or ET-1 (10(-9) M) on the activation of either pathway in normal or high glucose conditions. To determine if there was altered vascular reactivity in intact vessels we tested the effects of 5-HT and ET-1 interaction using myographs to measure isometric contractions of rat thoracic aortic rings. 5-HT (10(-9) M) and ET-1 (10(-12) M) stimulate enhanced contractile responses to each other that were inhibited by JAK2 and p42/44 MAPK antagonists. Our findings demonstrate that both 5-HT and ET-1 at physiological concentrations could interact with each other and activate p42/44 MAPK and JAK2 signaling pathways to cause an increase in smooth muscle contraction that could lead to altered vascular function.

A Mechanism Underlying Hypertensive Occurrence in the Metabolic Syndrome: Cooperative Effect of Oxidative Stress and Calcium Accumulation in Vascular Smooth Muscle Cells

Several lines of evidence indicate that reactive oxygen species (ROS) overproduction under the metabolic syndrome condition is the leading cause of cardiovascular events. Calcium is an important stimulus for vasoconstriction and plays a pivotal role in the development of hypertension. Here, we investigate whether a relationship exists between metabolic syndrome-induced mitochondrial ROS overproduction and Ang II-mediated Ca2+ release in vascular smooth muscle cells (VSMC). The effect of mitochondrial ROS on AT1 expression, and Ca2+ and IP3 generation was studied in 2 VSMC models of metabolic syndrome using fura-2/AM probes and ELISA-based assay. Ang II-mediated aortic ring contraction in SD rats fed with high-fat diet (HFD) was measured using a force transducer connected to chart recorder. In the metabolic syndrome, almost 2-fold increased mitochondrial O2 - significantly upregulated AT1 expressions by ~60%, companied by elevated Ca2+ and IP3 levels in VSMC and enhanced aortic rings contraction. All these increments were blocked by rotenone (inhibitor of mitochondrial respiratory chain complex I), ruthenium red (inhibitor of calcium uniporter), cyclosporin A (inhibitor of mitochondrial permeability pore), and N-acetylcysteine. Therefore, in the states of metabolic syndrome, ROS overproduction in mitochondrial complex I enhances Ang II-mediated vascular contraction via an AT1-dependent pathway. In addition, the import of Ca2+ from endoplasmic reticulum to mitochondria via calcium uniporter and mitochondrial permeability pore seems to serve as a mechanism to further aggravate mitochondrial damage and vascular dysfunction that may contribute to the occurrence of hypertension.

Pharmacological characterization of FE 201874, the first selective high affinity rat V1A vasopressin receptor agonist

Distinct vasopressin receptors are involved in different physiological and behavioural functions. Presently, no selective agonist is available to specifically elucidate the functional roles of the V1A receptor in the rat, one of the most widely used animal models. FE 201874 is a new derivative of the human selective V1A receptor agonist F180. In this study, we performed a multi-approach pharmacological and functional characterization of FE 201874 to determine whether it is selective for V1A receptors. We modified an available human selective V1A receptor agonist (F180) and determined its pharmacological properties in cell lines expressing vasopressin/oxytocin receptors (affinity and coupling to second messenger cascades), in an ex vivo model (aorta ring contraction) and in vivo in rats (proliferation of adrenal cortex glomerulosa cells and lactation). FE 201874 exhibited nanomolar affinity for the rat V1A receptor; it was highly selective towards the rat V1B and V2 vasopressin receptors and behaved as a full V1A agonist in all the pharmacological tests performed. FE 201874 bound to the oxytocin receptor, but with moderate affinity, and behaved as an oxytocin antagonist in vitro, but not in vivo. On functional grounds, all the data demonstrate that FE 201874 is the first selective agonist of the rat V1A receptor isoform available. Hence, FE 201874 may have potential as a treatment for the vasodilator-induced hypotension occurring in conditions such as septic shock and could be the most suitable compound for discriminating between the behavioural effects of arginine vasopressin and oxytocin.

앵엽(櫻葉) 에탄올 추출물의 혈관이완 효능 및 작용기전에 대한 연구

Objectives : The purpose of present study was to investigate the vasorelaxant activities and mechanisms of action of the ethanol extract of P. yedoensis leaf (PYL) on isolated rat aortic rings. Methods : Dried P. yedoensis leaves were extracted 3 times with 100% ethanol for 3 h in a reflux apparatus. Isolated rat aortic rings were suspended in organ chambers containing 10 ml Krebs-Henseleit (K-H) solution. The rings were maintained at <TEX>$37^{\circ}C$</TEX> and aerated with a mixture of 95% <TEX>$O_2$</TEX> and 5% <TEX>$CO_2$</TEX>. Changes in their tension were recorded via isometric transducers connected to a data acquisition system. Results : PYL relaxed the contraction of aortic rings induced by phenylephrine (PE, 1 <TEX>${\mu}M$</TEX>) or KCl (60 mM) in a concentration dependent manner. However, the vasorelaxant effects of PYL on endothelium-denuded aortic rings were lower than endothelium-intact aortic rings. And the vasorelaxant effects of PYL on endothelium-intact aortic rings were reduced by pre-treatment with <TEX>$N{\omega}$</TEX>-Nitro-L-arginine methyl ester (10 <TEX>${\mu}M$</TEX>), methylene blue (10 <TEX>${\mu}M$</TEX>), 1-H-[1,2,4]-oxadiazolo-[4,3-<TEX>${\alpha}$</TEX>]-quinoxalin-1-one (10 <TEX>${\mu}M$</TEX>), tetraethylammonium (5 mM). In addition, PYL inhibited the contraction induced by extracellular <TEX>$Ca^{2+}$</TEX> in endothelium-denuded aortic rings pre-contracted by PE or KCl in <TEX>$Ca^{2+}$</TEX>-free K-H solution. Conclusions : These results suggest that PYL exerts its vasorelaxant effects via the activation of Nitric Oxide (NO) formation by means of L-arginine and NO-cGMP pathways and via the blockage of receptor operated calcium channels, voltage dependent calcium channels and calcium-activated potassium channels.