Contraction Of Aorta Research Articles

Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase

This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nω-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).

Vascular smooth muscle cell contraction and relaxation in the isolated aorta: a critical regulator of large artery compliance.

Over the past few decades, isometric contraction studies of isolated thoracic aorta segments have significantly contributed to our overall understanding of the active, contractile properties of aortic vascular smooth muscle cells (VSMCs) and their cross‐talk with endothelial cells. However, the physiological role of VSMC contraction or relaxation in the healthy aorta and its contribution to the pulse‐smoothening capacity of the aorta is currently unclear. Therefore, we investigated the acute effects of VSMC contraction and relaxation on the isobaric biomechanical properties of healthy mouse aorta. An in‐house developed set‐up was used to measure isobaric stiffness parameters of periodically stretched (10 Hz) aortic segments at an extended pressure range, while pharmacologically modulating VSMC tone and endothelial cell function. We found that the effects of α1‐adrenergic stimulation with phenylephrine on the pressure‐stiffness relationship varied in sensitivity, magnitude and direction, with the basal, unstimulated NO production by the endothelium playing a pivotal role. We also investigated how arterial disease affected this system by using the angiotensin‐II‐treated mouse. Our results show that isobaric stiffness was increased and that the aortic segments demonstrated a reduced capacity for modulating the pressure‐stiffness relationship. This suggests that not only increased isobaric stiffness at normal pressure, but also a reduced capacity of the VSMCs to limit the pressure‐associated increase in aortic stiffness, may contribute to the pathogenesis of this mouse model. Overall, this study provides more insight in how aortic VSMC tone affects the pressure‐dependency of aortic biomechanics at different physiological and pathological conditions.

Inhibition of TMEM16A by Docosahexaenoic Acid Plays a Crucial Role in Blood Vessel Relaxation

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid with beneficial cardiovascular effects, such as vasodilation. The mechanism underlying this vasodilation, however, is not fully defined. In arterial smooth muscle, TMEM16A chloride channels promote membrane depolarisation and vessel contraction. Cloned TMEM16A channels are inhibited by DHA. Here, we examine whether inhibition of native TMEM16A by DHA plays a role in aortic relaxation. Patch-clamp electrophysiology and isometric tension recordings were utilised to study the effect of DHA on cloned TMEM16A channels and aortic contractility. DHA inhibited TMEM16A currents in a dose dependent manner, IC50=15.4±1.5 μM (n=10). To study the regulation of TMEM16A by DHA in response to agonist-induced increase in [Ca2+]i, TMEM16A was co-expressed with the α1A-adrenoreceptor and currents recorded in response to 1 μM phenylephrine (PE). In the absence and presence of DHA (10 μM) the TMEM16A current increased by 7.6±1.8 fold (n=12) and 2.8±0.5 fold (n=6), respectively (p<0.05). In aortic rings, inhibition of TMEM16A with either 70 μM DHA or 1 μM 2-(4-Chloro-2-methylphenoxy)-N′-(2-methoxybenzylidene)acetohydrazide (Ani9) reduced PE-induced aortic contraction by 1.9±0.4 fold (n=9) and 2.0±0.4 fold (n=7), respectively. Inhibition of the NKCC1 transporter with 40 μM bumetanide, to lower intracellular Cl- concentration ([Cl-]), reduced aortic response to 1 μM PE by 3.3±0.4 fold (n=10). Conversely, lowering extracellular [Cl-] increased the aortic response to 1 μM PE by 9.3±3.4 fold (n=5); this effect was reduced to 2.8±1.0 fold (n=5) in the presence of 70 μM DHA (p<0.05), implicating TMEM16A channels in the response. DHA-mediated inhibition of TMEM16A channels reduces aortic contractility. This study highlights TMEM16A as a target for modulating vascular tone. Identification of the TMEM16A DHA binding site may enable development of new small molecules aiming to reduce blood pressure.

Altered Purinergic Receptor Sensitivity in Type 2 Diabetes-Associated Endothelial Dysfunction and Up₄A-Mediated Vascular Contraction.

Purinergic signaling may be altered in diabetes accounting for endothelial dysfunction. Uridine adenosine tetraphosphate (Up4A), a novel dinucleotide substance, regulates vascular function via both purinergic P1 and P2 receptors (PR). Up4A enhances vascular contraction in isolated arteries of diabetic rats likely through P2R. However, the precise involvement of PRs in endothelial dysfunction and the vasoconstrictor response to Up4A in diabetes has not been fully elucidated. We tested whether inhibition of PRs improved endothelial function and attenuated Up4A-mediated vascular contraction using both aortas and mesenteric arteries of type 2 diabetic (T2D) Goto Kakizaki (GK) rats vs. control Wistar (WT) rats. Endothelium-dependent (EDR) but not endothelium-independent relaxation was significantly impaired in both aortas and mesenteric arteries from GK vs. WT rats. Non-selective inhibition of P1R or P2R significantly improved EDR in aortas but not mesenteric arteries from GK rats. Inhibition of A1R, P2X7R, or P2Y6R significantly improved EDR in aortas. Vasoconstrictor response to Up4A was enhanced in aortas but not mesenteric arteries of GK vs. WT rats via involvement of A1R and P2X7R but not P2Y6R. Depletion of major endothelial component nitric oxide enhanced Up4A-induced aortic contraction to a similar extent between WT and GK rats. No significant differences in protein levels of A1R, P2X7R, and P2Y6R in aortas from GK and WT rats were observed. These data suggest that altered PR sensitivity accounts for endothelial dysfunction in aortas in diabetes. Modulating PRs may represent a potential therapy for improving endothelial function.

Effects of Cinnamomum cassia extract on oxidative stress, immunreactivity of iNOS and impaired thoracic aortic reactivity induced by type II diabetes in rats

Type II diabetes is known to cause neuropathy, nephropathy and retinopathy. However, cardiovascular disorders associated with diabetes have been ignored. In traditional medicine, cinnamon (Cinnamomum cassia) bark has been used for its abilities to relieve fever, inflammation and chronic bronchitis. In the present study, the effect of Cinnamomum cassia extract (CN) on the thoracic aorta in an experimental type II diabetes model was investigated. In rats administered with nicotinamide + streptozotocin, significant endothelial dysfunction and oxidative stress were characterised by increased inducible nitric oxide synthase (iNOS) and decreased insulin/proinsulin levels. This impairment was prevented by administering 1000 mg/kg metformin or 500-1000-1500 mg/kg CN. CN administration attenuated the inflammatory response by decreasing the levels of malondialdehyde (MDA), Nitric oxide (NO) and increasing Glutathione peroxidase (GPx), glutathione (GSH). In addition, CN administration was shown to cause down-regulating effects on iNOS in thoracic aorta. These findings reveal that CN could prevent chronic complications of experimentally induced type II diabetes by attenuating inflammation, oxidant/antioxidant imbalance, and normalised contraction and relaxion responses in the thoracic aorta.

Enhanced calcium entry via activation of NOX/PKC underlies increased vasoconstriction induced by methylglyoxal

Advanced glycation end-products (AGEs) play a pivotal role in macro- and micro-vascular diabetic complications. We investigated the mechanism by which methylglyoxal (an endogenous generator of AGEs) affects vascular contractility using the isolated artery technique. Contractile responses to vasoconstrictors phenylephrine (PE), angiotensin II (Ang II), vasopressin (VP) and KCl were measured in the isolated rat aorta following one-our exposure to methylglyoxal (50–200 μM). The perfused rat kidney was employed to confirm the effect of methylglyoxal on microvessels. Methylglyoxal-induced changes in cytosolic calcium were measured in the smooth muscle layer of the aorta with the calcium-sensing fluorophore Fluo-4 AM. Methylglyoxal significantly increased maximal contraction of the rat aorta to PE, Ang II and VP. Similar results were seen in response to the depolarizing vasoconstrictor KCl in macro and micro vessels. The methylglyoxal-induced increases in aortic contraction mediated by the agonist and KCl were endothelium independent. Methylglyoxal-induced increases in KCl-dependent aortic contraction were abolished after the removal of extracellular calcium or in the presence of the calcium channel blocker nifedipine. Incubation with the antioxidant N-acetyl-l-cysteine (NAC), apocynin (a nonselective NADPH oxidase (NOX) inhibitor) or chelerythrine (a protein kinase C (PKC) inhibitor) prior to methylglyoxal pre-treatment reversed the methylglyoxal-induced increases in the rat aortic contractility. In conclusion, the formation of AGEs increases vasoconstriction of both macro- and micro-vessels by increasing the voltage-activated calcium entry in vascular smooth muscles in a NOX and PKC dependent manner.

Enhanced A1 adenosine receptor-induced vascular contractions in mesenteric artery and aorta of in L-NAME mouse model of hypertension

L-NAME-induced hypertension is commonly used to study endothelial dysfunction and related vascular effects. It has been reported that genetic deletion of A1 adenosine receptor (AR) reduces blood pressure (BP) increases in mice and thus, suggesting the involvement of A1AR. Thus, we sought to determine whether A1AR-induced vascular responses were altered in this mouse model of hypertension. L-NAME (1 mg/ml) was given in the drinking water for 28 days to mice. The BP was monitored using non-invasive tail-cuff system. Muscle tension studies were performed using DMT for mesenteric arteries (MAs) and organ bath for aorta. Protein expression was analyzed by western blot. Significantly, higher systolic and mean arterial blood pressure was noted in L-NAME mice. In MAs, higher 2-Chloro-N6-cyclopentyladenosine (CCPA, selective A1AR agonist) induced contractions in hypertensive mice were observed. This enhanced contraction was inhibited by HET0016 (Cytochrome 450 4A inhibitor, 10 µM, 15 min). Contrary, 5′-(N-Ethylcarboxamido) adenosine (NECA, non-selective AR agonist) induced vascular responses were comparable in both groups. Pinacidil (KATP channel opener) induced relaxation was significantly increased in hypertensive mice. In aorta, CCPA-induced contractions were enhanced and inhibited by HET0016 in hypertensive mice. Notably, NECA-induced contractions in aorta were enhanced in hypertensive mice. Higher expressions of A1AR and Cyp4A were noted in MAs of hypertensive mice. In addition, in aorta, higher A1AR and comparable Cyp4A levels were observed in hypertensive mice. A1AR-induced vascular contractions were enhanced in hypertensive mice aorta and MAs. Cyp4A plays a role in altered vascular responses in MAs.

Vasorelaxant effect of the Lippia alba essential oil and its major constituent, citral, on the contractility of isolated rat aorta.

The Lippia alba (Mill.) N.E. Brown (Verbenaceae) species popularly known as lemon balm has sedative, analgesic and spasmolytic properties. This study aimed to evaluate the vasorelaxant effect of the L. alba essential oil (EOLa) and its major constituent, citral, rat on aorta. Isometric muscle contraction were induced by potassium (K 60 mM) or phenylephrine (PHE, 0.1 μM) in isolated aortic rings. EOLa and citral promoted a smooth muscle relaxant action, which was potentiated by the presence of the endothelium; PHE-induced contractions (0.1 μM) in aorta with endothelium, had EC50 values of 352.73 ± 19.39 μg/mL and 99.34 ± 7.2 μg/mL for EOLa and citral, respectively. In the presence of a nitric oxide synthase inhibitor, L-NAME, the EC50 values were 654.19 ± 10.46 μg/mL and 601.66 ± 10.922 μg/mL for EOLa and citral, respectively. EOLa and citral dose-dependently relaxed contractions induced by BAY-K 8644, a calcium channel agonist, and by Phorbol 12,13-dibutyrate an activator of protein kinase C. EOLa and citral produced a vasorelaxant effect in isolated aorta which was potentiated by the presence of endothelium. In summary, EOLa and citral, probably using several mechanisms of action, relaxed aortic smooth muscle with maximal pharmacologic efficacy.

Electrical field-induced contractions on Crotalus durissus terrificus and Bothrops jararaca aortae are caused by endothelium-derived catecholamine.

Endothelium is the main source of catecholamine release in the electrical-field stimulation (EFS)–induced aortic contractions of the non- venomous snake Panterophis guttatus. However, adrenergic vasomotor control in venomous snakes such as Crotalus durissus terrificus and Bothrops jararaca has not yet been investigated. Crotalus and Bothrops aortic rings were mounted in an organ bath system. EFS-induced aortae contractions were performed in the presence and absence of guanethidine (30 μM), phentolamine (10 μM) or tetrodotoxin (1 μM). Frequency-induced contractions were also performed in aortae with endothelium removed. Immunohistochemical localization of both tyrosine hydroxylase (TH) and S-100 protein in snake aortic rings and brains, as well as in human tissue (paraganglioma tumour) were carried out. EFS (4 to 16 Hz) induced frequency-dependent aortic contractions in both Crotalus and Bothrops. The EFS-induced contractions were significantly reduced in the presence of either guanethidine or phentolamine in both snakes (p<0.05), whereas tetrodotoxin had no effect in either. Removal of the endothelium abolished the EFS-induced contractions in both snakes aortae (p<0.05). Immunohistochemistry revealed TH localization in endothelium of both snake aortae and human vessels. Nerve fibers were not observed in either snake aortae. In contrast, both TH and S100 protein were observed in snake brains and human tissue. Vascular endothelium is the main source of catecholamine release in EFS-induced contractions in Crotalus and Bothrops aortae. Human endothelial cells also expressed TH, indicating that endothelium- derived catecholamines possibly occur in mammalian vessels.

Abstract P145: The Bacterial Metabolite Trimethylamine (TMA), but Not Trimethylamine N-Oxide (TMAO), Causes Vascular Contraction

Circulating levels of TMAO are elevated in humans with cardiometabolic diseases. The gut microbiota-initiated TMA/flavin-containing monooxygenase 3 (FMO3)/TMAO pathway has been identified as a potential modulator of host cardiometabolic phenotypes. Like TMA/TMAO, FMO3 expression is positively correlated with body mass index and high-fat diet-induced obesity We hypothesized that TMA/TMAO stimulate vascular contraction which could be modified by perivascular adipose tissue (PVAT) expression of FMO3. Our model was the isolated thoracic aorta with (+) or without (-) PVAT, in presence (+) or in absence (+) of endothelium (E) of male Sprague Dawley rat to perform tissue bath studies to measure isometric tone using a large range of TMA/TMAO concentrations: 1 nM-10 -1 M. Immunohistochemistry (IHC) studies were done to identify the presence of FMO3 in aorta. TMA and TMAO, at any concentration, did not relax half maximally contracted tissues when compared to vehicle control. TMA and TMAO elicited arterial contraction independent of PVAT or E status. Interestingly, contractions stimulated by TMA were significantly higher (% PE max 123.7±15.6%) than by TMAO (% PE max 38.3±15.5%). IHC studies revealed FMO3 in aortic PVAT, but the FMO3 inhibitor methimazole (100 μM) did not affect aortic contraction stimulated by TMA (+PVAT). The L type Ca2+ channel antagonist nifedipine (100 nM), reduced the TMA-induced contraction (% PE max: +PVAT: 73±9%; -PVAT: 63±6.7%), compared to vehicle (+PVAT: 149.8±18%; -PVAT: 111.8±9%). These results support that TMA can induce arterial contraction with higher potency and efficacy compared to TMAO, independent of the conversion to TMAO. The contractile mechanism is ≈ 50% dependent of Ca 2+ influx through L-type VOC. Considering the high concentration needed to achieve the contraction it is unlikely that increasing vascular tone is a mechanism that could contribute to CVD but dependence of contraction on Ca 2+ channel activation suggests specific activity of TMA in the vessel wall that warrants further study.

Farnesoid X Receptor Activation Modulates Calcium Homeostasis in Rat Aortic Vascular Smooth Muscle Cells.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is expressed in the vasculature; our previous work showed that FXR regulated vascular reactivity through NO mechanism. The underlying mechanism for the regulation of vascular tension by FXR remains unclear. The present work was designed to investigate whether FXR regulates calcium homeostasis in aortic vascular smooth muscle cells (VSMCs). Protein abundances of angiotensin II type 1 and 2 receptors (AT₁R, AT₂R), bradykinin type 1 and 2 receptors (B₁R, B₂R), and the inositol 1,4,5-trisphosphate receptor (IP₃R) were analyzed by western blotting. Kallikrein activity and bradykinin content were assayed by using spectrophotometry and a bradykinin assay kit, respectively. Aortic contraction, intracellular Ca²⁺ concentrations ([Ca²⁺]i), sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) activities, and IP₃-evoked Ca²⁺ release were investigated, following FXR activation in the presence or absence of AT₂R and B₂R blockade. We found that the FXR agonists GW4064 and INT-747 increased the protein abundance of AT₂R and B₂R in rat aortic VSMCs. AT₂R blockade with PD123319 reversed the effects of FXR agonists on kallikrein activity, B₂R, and bradykinin levels. Moreover, we found that GW4064 and INT-747 decreased intracellular [Ca²⁺], increased SERCA activity, downregulated IP₃R₁ expression, and attenuated IP₃-induced Ca²⁺ release. These effects were partially reversed by AT₂R and B₂R blockade with PD123319 and HOE140, respectively. Our data suggest that FXR regulates vascular tension by modulating extracellular Ca²⁺ influx and intracellular Ca²⁺ release from the sarcoplasmic reticulum via activation of an AT₂R-B₂R pathway in rat aortic VSMCs.

Perivascular adipose tissue protects against the vascular dysfunction induced by acute ethanol intake: Role of hydrogen peroxide

AimWe investigated the consequences of acute ethanol intake on the anti-contractile effect of perivascular adipose tissue (PVAT). MethodsThe effects of a single dose of ethanol (1 g/kg; p.o. gavage) on the vascular function were assessed within 30 min in male Wistar rats. ResultsEthanol decreased the relaxation induced by acetylcholine and increased the contraction induced by phenylephrine in endothelium-intact, but not in endothelium-denuded aortas without PVAT. The vascular dysfunction induced by ethanol was not observed in aortic rings with PVAT. Nω-Nitro-l-arginine methyl ester (L-NAME), NG-nitro-l-arginine (L-NNA) and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), but not tiron or tempol, increased the contraction induced by phenylephrine in endothelium-intact aortas with PVAT from control and ethanol-treated rats. Catalase increased phenylephrine-induced contraction in aortas with PVAT from ethanol-treated rats, but not from control rats. Conversely, inhibition of catalase with aminotriazole decreased phenylephrine-induced contraction in aortas from ethanol-treated rats. Treatment with ethanol increased hydrogen peroxide (H2O2) levels and decreased catalase activity in aortas with PVAT. Ethanol increased superoxide anion (O2−) generation in aortas with or without PVAT. Superoxide dismutase (SOD) activity was not affected by ethanol intake. In situ quantification of H2O2 using 2′7′dichlorodihydrofluorescein diacetate (DCFH-DA) revealed increased levels of H2O2 in periaortic PVAT from ethanol-treated rats. However, in situ evaluation of nitric oxide (NO) in both aorta and PVAT showed no differences between groups. ConclusionsOur study provides novel evidence that the periaortic PVAT protects against the vascular dysfunction induced by acute ethanol intake through a mechanism that involves increased generation of H2O2.

A Simple Method for Normalization of Aortic Contractility

Vascular contractile function changes in proliferative vascular diseases, e.g. atherosclerosis, and is documented using isolated blood vessels; yet, many laboratories differ in their approach to quantification. Some use raw values (e.g., mg, mN); others use a “percentage of control agonist” approach; and others normalize by blood vessel characteristic, e.g. length, mass, etc. A lack of uniformity limits direct comparison of contractility outcomes. To address this limitation, we developed a simple 2-step normalization method: (1) measure blood vessel segment length (mm), area (mm²) and calculate volume (mm³); then, (2) normalize isometric contraction (mN) by segment length and volume. Normalized aortic contractions but not raw values were statistically different between normal chow and high-fat diet-fed mice, supporting the practical utility and general applicability of normalization. It is recommended that aortic contractions be normalized to segment length and/or volume to reduce variability, enhance efficiency, and to foster universal comparisons across isometric myography platforms, laboratories, and experimental settings.

Electrical field stimulation-induced contractions on Pantherophis guttatus corpora cavernosa and aortae.

A tetrodotoxin (TTX)-resistant mechanism is responsible for the electrical field stimulation (EFS)-induced contractions and relaxations of Crotalus durissus terrificus corpora cavernosa. Here it was investigated whether this mechanism also occurs in corpora cavernosa and aortae of the non-venomous snake Pantherophis guttatus corpora cavernosa and aortae. Corpora cavernosa and aortic rings isolated from Pantherophis guttatus snake were mounted in organ bath system for isometric tension recording. EFS-induced contractions in both tissues were performed in the presence and absence of guanethidine (30 μM), phentolamine (10 μM) and tetrodotoxin (1 μM). In another set of experiments, the endothelium was removed from aortic rings and EFS-induced contractions were performed in the denuded rings. Electrical field stimulation-induced contractions were frequency-dependent in Pantherophis guttatus corpora cavernosa and aortic rings. The contractions were significantly reduced in the presence of guanethidine (30 μM) or phentolamine (10 μM). Pre-treatment with tetrodotoxin had no effect on the EFS-induced contractions of either corpora cavernosa or aortic rings. Surprisingly, the EFS-induced contractions of aortic rings denuded of endothelium were almost abolished. These results indicate that the TTX-resistant mechanism is present in EFS-induced contractions of Pantherophis guttatus corpora cavernosa and aortae. The experiments performed in the aorta indicate that the endothelium is the main source for the release of catecholamines induced by EFS.

Selectivity of Ca2+ channel blockers for dilator actions on the isolated lower esophageal sphincter and aorta from rats

We compared dilator actions of representative four Ca2+ channel blockers on the isolated lower esophagus sphincter (LES) and thoracic aorta from rats. Verapamil, diltiazem, nifedipine and cilnidipine suppressed KCl-induced contractions of LES and thoracic aorta in a concentration-dependent manner. The order of selectivity for LES, which was calculated as ratio of IC50 value for thoracic aorta divided by that for LES, was diltiazem > verapamil > nifedipine > cilnidipine. These results suggest that diltiazem more preferentially dilates the LES whereas cilnidipine is expected to have lower potential risk of gastroesophageal dysfunction during the antihypertensive therapy.

Ergothioneine prevents endothelial dysfunction induced by mercury chloride.

Exposure to mercury has detrimental effects on the cardiovascular system, particularly the vascular endothelium. The present study aimed to investigate the effects of ergothioneine (EGT) on endothelial dysfunction induced by low-dose mercury chloride (HgCl2). Agonist-induced contractions and relaxations were evaluated in isolated aortic rings from 3-month-old male Wistar rats treated by intra-muscular injection to caudal hind leg muscle with HgCl2 (first dose, 4.6 µg/kg; subsequent doses, 0.07 µg/kg/day for 15 days) and optionally with EGT (2 µg/kg for 30 days). Reactive oxygen species (ROS) in aortic rings were measured by means of lucigenin- and luminol-enhanced chemiluminescence. The protein level of endothelial nitric oxide synthase was evaluated by ELISA. Blood glutathione (GSH) and catalase levels, lipid peroxidation and total nitrite were measured spectrophotometrically. The results indicated that low-dose HgCl2 administration impaired acetylcholine (ACh)-induced relaxation and potentiated phenylephrine- and serotonin-induced contractions in rat aortas. In addition, HgCl2 significantly increased the levels of ROS in the aortic tissue. EGT prevented the loss of ACh-induced relaxations and the increase in contractile responses. These effects were accompanied by a significant decrease in ROS levels. EGT also improved the ratio of reduced GSH to oxidized GSH and catalase levels with a concomitant decrease in lipid peroxidation. In conclusion, to the best of our knowledge, the present study was the first to report that EGT prevents endothelial dysfunction induced by low-dose HgCl2 administration. EGT may serve as a therapeutic tool to reduce mercury-associated cardiovascular complications via improving the antioxidant status.

Roles and mechanism of protein kinase D in vasoconstriction

Protein kinase D (PKD) is known to be implicated in a variety of cellular processes and physiological functions, including cell motility, cardiac contraction, angiogenesis and smooth muscle contraction through regulation of multiple molecules. Although these regulations are presumed to be closely associated with vasomotor activity, role of PKD in vascular function is unknown. Then, we investigated the role of PKD in systemic circulation and contraction of aorta in in rats. We also investigated precise role s of PKD isoforms using human aortic smooth muscle cells (HASMCs) with RNA interference.Material and methodsSystemic blood pressure (SBP) through femoral artery, cardiac output (CO) and systemic vascular resistances (SVR) were measured in Sprague‐Dawley rats. The hemodynamic changes in response to intravenous injections of PKD inhibitor, CRT0066101 were recorded. Isometric tension of aorta ring samples was measured to examine inhibitory effect of CRT0066101 on norepinephrine (NE)‐induced contraction. To explore mechanisms of PKD in vasomotor activity, phosphorylations of PKD, myosin targeting subunit‐1 (MYPT1), CPI‐17, regulatory myosin light chain (RLC) and heat shock protein 27 (HSP27) stimulated with NE were measured as an index of activation of the pathways in aorta rings. In HASMCs, simultaneous measurements of intracellular calcium concentrations and cell shortening with NE stimulation were performed. PKD1, 2, and 3 were knocked down by their specific siRNA respectively in HASMCs for measurements of phosphorylation of PKD, MYPT1, CPI‐17 and RLC and actin polymerization.ResultsInjection of CRT0066101 decreased SBP and SVR. CRT0066101 also inhibited NE‐induced contraction in aorta rings dose‐dependently. In the rings, NE increased phosphorylations of PKD, MYPT1, RLC and CPI‐17, which were attenuated by pretreatment of CRT0066101 except CPI‐17. NE did not increase HSP27 phosphorylation in the rings. In HASMCs, CRT0066101 inhibited NE‐induced cell shortening without affecting increased calcium concentrations. In HASMCs, increased phosphorylations of MYPT1 and RLC with NE were significantly inhibited by PKD1 but not PKD2, 3 knock down. NE did not affect actin polymerization in the cells. Collectively, attenuation of NE‐induced contraction of aorta rings with CRT0066101 was associated with decreased phosphorylations of MYPT1 and RLC, but not CPI‐17 and HSP27. In HASMCs, PKD1 knocked down or PKD inhibition attenuated NE‐induced phosphorylation of MYPT1 and RLC without affecting actin polymerization and intracellular calcium regulation.ConclusionsPKD1 may play roles in contraction of aorta and systemic circulation. The regulation of vasomotor activity by PKD1 may be associated with phosphorylation of MYPT1. Understanding of PKD may lead to novel regulation of circulation.Support or Funding InformationThis work was supported by Grant‐in‐Aid for Scientific Research (C) 17K11058 from Japan Society for the Promotion of ScienceThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Xyloketal B exerts antihypertensive effect in renovascular hypertensive rats via the NO-sGC-cGMP pathway and calcium signaling.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. (No 2508). We previously showed that Xyl-B promoted endothelial NO release and protected against atherosclerosis through the Akt/eNOS pathway. Vascular NO production regulates vasoconstriction in central and peripheral arteries and plays an important role in blood pressure control. In this study, we examined whether Xyl-B exerted an antihypertensive effect in a hypertensive rat model, and further explored the possible mechanisms underlying its antihypertensive action. Administration of Xyl-B (20 mg·kg-1·d-1, ip, for 12 weeks) significantly decreased the systolic and diastolic blood pressure in a two-kidney, two-clip (2K2C) renovascular hypertensive rats. In endothelium-intact and endothelium-denuded thoracic aortic rings, pretreatment with Xyl-B (20 μmol/L) significantly suppressed phenylephrine (Phe)-induced contractions, suggesting that its vasorelaxant effect was attributed to both endothelial-dependent and endothelial-independent mechanisms. We used SNP, methylene blue (MB, guanylate cyclase inhibitor) and indomethacin (IMC, cyclooxygenase inhibitor) to examine which endothelial pathway was involved, and found that MB, but not IMC, reversed the inhibitory effects of Xyl-B on Phe-induced vasocontraction. Moreover, Xyl-B increased the endothelial NO bioactivity and smooth muscle cGMP level, revealing that the NO-sGC-cGMP pathway, rather than PGI2, mediated the anti-hypertensive effect of Xyl-B. We further showed that Xyl-B significantly attenuated KCl-induced Ca2+ entry in smooth muscle cells in vitro, which was supposed to be mediated by voltage-dependent Ca2+ channels (VDCCs), and reduced ryanodine-induced aortic contractions, which may be associated with store-operated Ca2+ entry (SOCE). Taken together, these findings demonstrate that Xyl-B exerts significant antihypertensive effects not only through the endothelial NO-sGC-cGMP pathway but also through smooth muscle calcium signaling, including VDCCs and SOCE.

L-arginine and Arginase Products Potentiate Dexmedetomidine-induced Contractions in the Rat Aorta.

The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. L-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of L-arginine in modulating the overall vascular response to dexmedetomidine. Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 μM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), L-arginine, (S)-(2-boronethyl)-L-cysteine hydrochloride (arginase inhibitor), N-hydroxy-L-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed. Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with L-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous L-arginine augmented the dexmedetomidine-induced contractions in the presence of L-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-L-cysteine hydrochloride (Emax 16 ± 4%) and N-hydroxy-L-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as L-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by L-arginine treatment in the presence of L-NAME (N = 4). These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of dexmedetomidine by L-arginine depends on arginase activity and the production of urea and ornithine.

Characterization of vascular dysregulation in meriones shawi after high-calorie diet feeding

ABSTRACTThe present study was initiated to characterize vascular dysregulations (contraction and relaxation) associated with metabolic defects in Merions shawi, a rodent from the gerbillidae family, submitted to 12 weeks high-calorie diet. This diet induces a type 2 diabetes/metabolic syndrome phenotype with hypertension. In diabetic meriones, body weight increase was associated with hyperglycemia, increased insulinemia, and insulin resistance. Compared to lean meriones, diabetic meriones showed decreased aorta contraction to noradrenaline, which was normalized after NOS inhibition. Endothelium-dependent relaxation to carbachol was enhanced, while relaxing effects of the NO donor SNAP and of diazoxide were unchanged. Insulin-evoked relaxation was depressed in aorta from diabetic meriones, and L-arginine relaxed contracted arteries from diabetic meriones, but not from lean meriones. Urine NOX level and iNOS mRNA muscle expression were significantly higher in diabetic meriones compared to lean animals. These data strongly suggest that iNOS may have a pathogenic role in vascular dysfunction observed in diet-induced diabetic meriones.