Phenylephrine Concentration-response Curves Research Articles

Abstract P231: Lipoprotein Lipase Activation Is A Novel Anti-contractile Mechanism That Is Both Endothelium-dependent And Independent.

Risk factors such as hypertriglyceridemia and hypercholesterolemia are commonly attributed to the pathogenesis of atherosclerosis, however, lipoprotein metabolism is also associated with end organ damage and cardiovascular events in hypertensive patients. Lipoprotein lipase (LPL) is an important hydrolase for lipoprotein catabolism. Previous work from our lab has shown that LPL activator, NO-1886 (Ibrolipim), significantly decreased vascular contraction. Physiologically, endothelial function limits contractility and endothelial dysfunction is a hallmark of hypertension. Therefore, we hypothesized that LPL activation would cause endothelial-dependent hypocontractility. To test this hypothesis, mesenteric resistance arteries (MRA) were isolated from male Fischer 344 rats at 3 months of age (n=4-12) and mounted onto a wire myograph. Phenylephrine (PE) concentration-response curves were performed with and without LPL activator, NO-1886 (30 min). Some MRA were denuded of their endothelium or incubated with inhibitors of endothelium-dependent vasodilation. As expected, activating LPL exerted a significant anti-contractile effect in a concentration-dependent manner [KCl (%), Veh: 125±3 vs. NO-1886 (1 uM): 116.0±17 vs. NO-1886 (10 uM): 108±9* vs. NO-1886 (50 uM): 55±4* vs. NO-1886 (100 uM): 29±5*, *p<0.05 vs. Veh]. However, contrary to our hypothesis, denudation only partially restored contraction [KCl (%),Veh: 125±3 vs. NO-1886 (50 uM): 55±4* vs. NO-1886+denuded: 76±4* # , *p<0.05 vs. Veh, # p<0.05 vs. NO-1886]. This suggests that there is both an endothelium-dependent and -independent mechanism by which LPL limits contractility. Endothelial-dependent hypocontractility was found to be mediated by nitric oxide synthase [KCl (%),Veh: 125±3 vs. NO-1886 (50 uM): 55±4 vs. NO-1886+L-NAME (100 uM): 90±5* # , *p<0.05 vs. Veh, # p<0.05 vs. NO-1886]. Endothelium-independent mechanisms by which NO-1886 causes hypocontractility have yet been clarified (data not shown). Overall, this suggests that LPL has an important physiological role in controlling vascular contraction through both endothelial-dependent and -independent means and loss of LPL activity could be a novel mechanism of endothelial dysfunction in hypertension.

Lipoprotein lipase underlies the vascular anti-contractile effect of perivascular adipose tissue (PVAT)

Physiologically, perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the vasculature. This effect is mediated by the biosynthesis and liberation of a myriad of anti-contractile molecules (e.g., adipokines). However, this anti-contractile function generally is lost with aging and cardiovascular diseases such as hypertension and diabetes. Lipoprotein lipase (LPL) is an enzyme classically known for hydrolyzing triglycerides into free fatty acids and glycerol. However, it is unknown whether LPL underlies the anti-contractile effect of PVAT. Specifically, we hypothesized that by inhibiting lipoprotein lipase (LPL) activity with Atglistatin, we would abolish the anti-contractile effect of PVAT. To test this hypothesis, mesenteric resistance arteries (MRA), with and without PVAT, were isolated from 3-month-old male Wistar rats and mounted on a wire myograph. Phenylephrine concentration-response curves were performed with and without Atglistatin (10 -5 M). As expected, PVAT exerted a significant anti-contractile effect on phenylephrine-induced contraction [Emax (mN), -PVAT-vehicle: 18.1±3.0 vs. +PVAT-vehicle: 7.1±1.1, p<0.05]. Incubation of MRA with intact PVAT with Atglistatin significantly increased contraction [Emax (mN), +PVAT-vehicle: 7.1±1.1vs. +PVAT-Atglistatin: 22.6±4.2, p<0.05], suggesting that LPL is indeed important for the anti-contractile effect of PVAT. Crucially, no difference was observed with Atglistatin in MRA lacking PVAT [Emax (mN), -PVAT-vehicle: 18.1±3.0 vs. -PVAT-Atglistatin: 18.5±2.9, p>0.05], indicating that Atglistatin was not increasing contraction via non-PVAT-dependent mechanisms. Overall, these data suggest that LPL is an important mechanism mediating the anti-contractile effect of PVAT and that reduced LPL activity could be contributing to pro-contractile PVAT in aging and cardiovascular diseases. NHLBI (R00HL151889), NIGMS (1P20GM103641 - Pilot Project), UofSC SOM (Startup funds) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lipoprotein lipase activation is a novel vascular anti-contractile mechanism that is lost in aging

Increasing incidence of cardiovascular events with age can be attributed to the natural deterioration in organ function, and manifests through a range of vascular phenotypes that include, endothelial dysfunction, hypercontractility, stiffening and remodeling, inflammation, and oxidative stress. Given the aging global population, the identification and translation of novel mechanisms and therapeutics will be essential to increase longevity and prevent premature death and disability. Physiologically, perivascular adipose tissue (PVAT) is known to impact vasculature by exerting an anti-contractile effect mediated by the biosynthesis and liberation of a myriad of anti-contractile molecules (e.g., adipokines). However, this anti-contractile function declines with age and in related diseases. Therefore, our study aimed to restore the anti-contractile effect of PVAT in arteries from aged animals. Specifically, we predicted that increasing lipoprotein lipase (LPL) activity would reinstate the anti-contractile effect of PVAT on aged arteries. To assess this hypothesis, mesenteric resistance arteries (MRA), with (+) and without (-) PVAT, were isolated from male Fisher 344 rats aged 3 (3M) or 16 months (16M) and mounted on a wire myograph. Phenylephrine (PE) concentration-response curves were measured in the presence or absence of LPL activator, NO-1886 (Ibrolipim, 10uM). As expected, PVAT exerted significant anti-contractile effect in MRA, regardless of age [PE 100uM (%), 3M (-PVAT): 112.4±13.3 vs. 3M (+PVAT): 27.6±28.5 {Figure 1.a.} and 16M (-PVAT): 115.4±23.1 vs. 16M (+PVAT): 35.0±34.1 {Figure 1.b.}]. Contrary to our hypothesis, NO-1886 had no effect on the anti-contractile effect of PVAT in 3M or 16M rats [PE 100uM (%), 3M (+PVAT +vehicle): 30.0±34.3 vs. 3M (+PVAT +NO-1886): 47.9±29.8 {Figure 1.c.} and 16M (+PVAT +vehicle): 48.6±42.3 vs. 16M (+PVAT +NO-1886): 60.0±36.9 {Figure 1.d.}]. Surprisingly, we observed an effect of NO-1886 in MRA in the absence of PVAT. Specifically, NO-1886 significantly decreased phenylephrine-induced contraction in 3M rats, but not 16M rats [PE 100uM (%), 3M (-PVAT +vehicle): 112.4±13.3 vs. 3M (-PVAT +NO-1886): 37.8±22.2 {Figure 1.e.} and 16M (-PVAT +vehicle): 115.4±23.1 vs. 16M (-PVAT +NO-1886): 103.1±23.1 {Figure 1.f.}]. Overall, this suggests that LPL has an important physiological role in controlling vascular contraction and this novel anti-contractile mechanism of LPL diminishes with biological age. Funding: NHLBI (R00HL151889), NIGMS (1P20GM103641 - Pilot Project), NIA (K01AG061263), UofSC SOM (Startup funds) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antagonism of α1-adrenoceptors by β3-adrenergic agonists: Structure–function relations of different agonists in prostate smooth muscle contraction

Effects of β3-adrenergic agonists on prostate smooth muscle contraction are poorly characterized, although mirabegron is used for treatment of lower urinary tract symptoms. Off-target effects of several β3-adrenergic agonists include antagonism of α1-adrenoceptors. Proposed, but unconfirmed explanations include phenylethanolamine backbones, found in some β3-adrenergic agonists and imparting interaction with catecholamine binding pockets of adrenoceptors. Here, we examined effects of β3-adrenergic agonists on contractions of human prostate tissues, including ZD7114 (without phenylethanolamine moiety), ZD2079 (phenylethanolamine backbone), BRL37344 and CL316243 (chloride-substituted phenylethanolamine deriatives). Prostate tissues were obtained from radical prostatectomy. Contractions by α1-adrenergic agonists and electric field stimulation (EFS) were studied in an organ bath. ZD7114 (10 µM) right-shifted concentration responses curves for α1-adrenergic agonists, resulting in increased EC50 values for phenylephrine, methoxamine and noradrenaline up to one magnitude, without affecting Emax values. ZD7114 (10 µM) inhibited EFS-induced contractions, resulting in reduced Emax values. All effects of ZD7114 were resistant to the β3-adrenergic antagonist L-748337, including increases in EC50 values for α1-adrenergic agonists, up to more than two magnitudes. Using 10 µM, neither ZD2079, BRL37344 or CL316243 affected α1-adrenergic or EFS-induced contractions. At escalated concentrations, BRL37344 (200 µM) right-shifted concentration response curves for phenylephrine, increased EC50 values for phenylephrine, and inhibited EFS-induced contractions, while CL316243 (300 µM) did not affect phenylephrine- or EFS-induced contractions. In conclusion, phenylethanolamine backbones are not decisive to impart α1-adrenoceptor antagonism to β3-agonists. Effects of β3-adrenergic agonists on prostate smooth muscle contraction are limited to off-target effects, including α1-adrenoceptor antagonism by ZD7114 and BRL37344.

PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α 1 -ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α 1 -BLOCKERS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (PD11)1 Sep 2021PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α1-ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α1-BLOCKERS Ru Huang, Yuhan Liu, Bingsheng Li, Ruixiao Wang, Alexander Tamalunas, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Ru HuangRu Huang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Bingsheng LiBingsheng Li More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001986.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Mirabegron inhibits detrusor smooth muscle contractions and is available for treatment of overactive bladder. Consequently, its effects on lower urinary tract symptoms (LUTS) suggestive of bengin prostatic hyperplasia (BPH) were subject of recent trials. However, available results are not yet conclusive, and effects of mirabegron on human prostate smooth muscle contraction are hardly characterized. Voiding symptoms suggestive of BPH may be driven by increased prostate smooth muscle tone. Here, we examined effects of mirabegron on contractions of human prostate tissues. METHODS: Human prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Comparisons were performed between whole groups (one-way ANOVA), and at single frequencies and agonist concentrations (multivariate analysis). RESULTS: Electric field stimulation (EFS) induced neurogenic, frequence-dependent contractions, while agonists induced concentration-dependent contractions. Mirabegron (10 µM) inhibited EFS-induced contractions (p <0.001 for controls vs. mirabegron between whole groups), which was observed at different frequencies and reflected by reduced Emax values calculated by curve fitting (contractions in controls 84±11% of KCl-induced contractions, but 14±2% after mirabegron, p <0.01). Miragebron increased the EC50 values for noradrenaline (controls 0.55±0.32 µM, mirabegron 5.1±2.4 µM, p <0.004), resulting in rightshifts of concentration response curves for noradrenaline-induced contractions (p <0.001) and in reduced contractions up to 10 µM noradrenaline, but in recovery at higher noradrenaline concentrations and unchanged Emax values. Similarly, EC50 values for the α1-adrenergic agonist phenylephrine were increased by mirabegron (controls 7.4±4.1 µM, mirabegron 29.2±8.9 µM, p=0.05), resulting in rightshifts of concentration response curves for phenylephrine (p <0.001) and in reduced contractions up to 30 µM phenylephrine, but in recovery at higher phenylephrine concentrations and in unchanged Emax values. Mirabegron slightly reduced contractions induced by the thromboxane A2 analog U46619 (p <0.03), without changing EC50 values. Contractions induced by endothelin-1 remained unchanged by mirabegron. CONCLUSIONS: Mirabegron inhibits neurogenic and α1-adrenergic smooth muscle contractions in the human prostate. Surprisingly, the pharmacologic pattern is similar to that of α1-blockers, including rightshifts of α1-adrenergic concentration response curves, reduced Emax values for neurogenic contractions, and lacking or small effects on non-adrenergic contractions. Source of Funding: China Scholarship Council (CSC) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e200-e200 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ru Huang More articles by this author Yuhan Liu More articles by this author Bingsheng Li More articles by this author Ruixiao Wang More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement Loading ...

Antihypertensive potential of selected pyrimidine derivatives: Explanation of underlying mechanistic pathways

This study was designed to determine the effectiveness of 5-(3-Hydroxybenzylidene)-2, 4, 6(1H, 3H, 5H)-pyrimidinetrione (SR-5), 5-(4-Hydroxybenzylidene)-2, 4, 6(1H, 3H, 5H)-pyrimidinetrione (SR-8), 5-(3-Chlorobenzylidene)-2, 4, 6(1H, 3H, 5H)-pyrimidinetrione (SR-9) and 5-(4-Chlorobenzylidene)-2, 4, 6(1H, 3H, 5H)-pyrimidinetrione (SR-10) against hypertension. In deoxycorticosterone acetate-salt rats, SR-5, SR-8, SR-9, and SR-10 reduced blood pressure and normalized renal functions. In isolated rat aortic rings, SR-5, SR-8, SR-9, and SR-10 relaxed phenylephrine (PE) and K+-induced contractions. The vasodilator effect was endothelium-independent. Test compounds caused a rightward shift of Ca++ and PE concentration-response curves with a reduction of maximum response. SR-5, SR-8, SR-9, and SR-10 inhibited PE peak contractions in a Ca++ free medium. In guinea-pig atria, SR5, SR-8, SR-9, and SR-10 caused a mild-to-moderate inhibition of force and rate of contractions. In the aorta and heart tissues, the test compounds enhanced glutathione-s-transferase, reduced glutathione and catalase levels, improved cellular architecture, and decreased lipid peroxidation and expression of inflammatory markers: cyclooxygenase 2, tumor necrosis factor alpha, phosphorylated c-Jun N-terminal kinase, and phosphorylated-nuclear factor kappa B, evidenced in the immunohistochemistry, enzyme-linked immunosorbent assay, western blot molecular investigations and a decreased mRNA expression of calcium channel in RT-PCR analysis. SR-5, SR-8, SR-9, and SR-10 increased the urinary output in rats and inhibited the human platelet aggregation. This study revealed that SR-5, SR-8, SR-9, and SR-10 possess BP lowering, reno-protective, vasodilatory (mediated via Ca++ antagonist, antioxidant and anti-inflammatory pathways), partial cardio-suppressant, diuretic, and antiplatelet effects, demonstrating their therapeutic potential in hypertension management.

Abstract 125: Low-Grade Chronic Infection Induces Vascular Dysfunction and Remodeling in Salt Sensitive Hypertension

According to the new AHA guidelines, over half of Americans have hypertension. Multiple factors contribute to hypertension including genetics, diet, stress, and infection. N-formyl peptides (NFPs) are a potent chemoattractant that bind formyl peptide receptors (FPR), an innate immune receptor. Interestingly, the only source of NFPs are bacteria and mitochondria, which evolved from bacteria. We have observed that FPR is expressed in arteries and its activation with bacterial (fMLPs) or mitochondrial (FMIT) NFPs induces vascular remodeling via actin polymerization. Given that increased gut permeability is present in hypertension, we questioned if bacterial NFPs derived from a leaky gut also play a role in hypertension. We hypothesized that bacterial NFPs activate FPR leading to vascular dysfunction and remodeling. We used male (6 weeks) Dahl Resistant (DR) and Sensitive (DS) rats on a low (0.3 %) or high salt (2 %) diet for 9 weeks. After 6 weeks some animals on high salt received water + antibiotic [neomycin (NEO), 0.5 g/L or amoxicillin (AMO), 50 mg/kg/day) for 3 weeks to decrease bacterial levels. Statistics: t-test *p&lt;0.05. We observed that FPR mRNA expression was increased in aorta from DS (low-salt) when compared to DR (low-salt) (DR: 0.96 ± 0.7 vs. DS: 1.3 ± 1.7*), suggesting that FPR is activated prior to the onset of hypertension. High-salt diet increased FPR mRNA expression in aorta from DR, but did not exacerbate it in DS compared to DS (low-salt). Phenylephrine and acetylcholine concentration-response curves (1nM – 1μM) were performed in mesenteric resistance arteries using wire myography. Due to increased arterial stiffness from severe hypertension in DS (high salt), these rats showed a decrease in contraction compared to DR. DS treated with NEO or AMO ameliorated this response (tension (mN): Emax: DS 11 ± 4 vs. DS+ NEO 17 ± 2*, S + AMO 17 ± 0.9*). No changes were observed in the relaxation curve. Using pressure myograph, it was observed that NEO and AMO reverted vascular remodeling [wall thickness (μm): DS 55 ± 4 vs. DS + NEO 38 ± 9* and DS + AMO 31± 9*; cross sectional area (μm 2 ): DS 45,624 ± 1370 vs. DS + NEO 28,700 ± 8370* and DS + AMO 23,276 ± 9775*]. We concluded that low-grade chronic infection plays a role in vascular dysfunction and remodeling observed in hypertension.

Effects of diosmin and crocin on metabolic syndrome-associated cardio-vascular complications in rats

This study aims to scrutinize the potential protective effect of diosmin and crocin against cardio-vascular complications attributed to metabolic syndrome (MS). For 16weeks, the rats were given 10% fructose in water and 3% salt in diet to induce MS over a 16-week period. At week 7 and for 10weeks, diosmin (50mg/kg, PO) or crocin (50mg/kg, PO) was administered daily. Non-invasive blood pressure (BP) was recorded in conscious animals. Thoracic aorta (macro vessels) and kidney (micro vessels) concentration-response curves for phenylephrine (PE) and acetylcholine (ACh) were analyzed. Electrocardiogram (ECG) parameters were recorded. Blood glucose level, serum insulin level, troponin T, advanced glycation end products (AGEs), malondialdehyde (MDA), and tumor necrosis factor-α (TNF-α) were measured. In addition, histological examination of aorta and heart tissues was performed. Diosmin and crocin alleviated cardio-vascular complications associated with MS. This is manifested in improvement of systolic and diastolic BP and ECG parameters. They ameliorated MS-induced exaggerated contractility to PE and improved the impaired dilatation to Ach in macro and micro vasculature. Diosmin and crocin reduced the elevated serum levels of insulin, AGEs, TNF-α, and MDA and reversed MS-induced structural changes of aorta and cardiac tissues. Diosmin and crocin offset the cardiac changes and vascular impairment associated with MS via attenuation of oxidative stress and suppression of inflammation.

499 Effect of norbornene derivative in corpus cavernosum relaxation, as 5-HT1A agonist and 5-HT2A antagonist

The aim of this study was to analyze the in vitro effects of Norbornene derivative (4-[3-[4-(o-methoxyphenyl)piperazin-1-yl]propoxy]-4-aza-tricyclo-[5.2.1.02,6]-dec-8-ene-3,5-dione; Nm), in the rat corpus cavernosum. All animal procedures and experimental protocols were approved by the Institutional Committee for Ethics in Animal Research/University of Campinas (protocol number: 4120-1). Male Wistar rats (6 months) were killed under isoflurane anesthesia and corpus cavernosum was isolated, assembled in an organ bath and suspended to an isometric force transducer. Concentration-response curves were obtained for Phenylephrine (PHE) and 5-Hydroxytryptamine (5-HT) (0.01 to 1 mM). Concentration-response curves to Prazosin (PZS) and Norbornene derivative (0.01 and 100 μM) were also performed, the tissues were pre-contracted by PE (3 μM) or 5-HT (10 μM) and the potency (pEC50) and maxima response (Emax) values were obtained. Data are expressed as mean + SEM, N= 4 in all experiments. The relaxation-induced by PZS and Nm showed Emax: 104.8%, pEC50: 7.25 ± 0.02 and Emax: 105.9%, pEC50: 6.60**, respectively, when pre-contracted by PHE. When pre-contracted by 5-HT, Nm showed Emax: 85.30%**, pEC50: 8.06 ± 0.11, and PZS Emax: 65.02%; pEC50: 7.82 ± 0.09. In single concentration tissues pre-contracted by PHE, PZS proved be more effective than Nm (Emax: 108.45% and 72.18%, respectively). In tissues pre-contracted with 5-HT, Nm proved to be more efficient than PZS (Emax: 92.77% and 68.71%, respectively). Moreover, a concentration-response curve of PHE was performed in the presence of Nm 100nM (Emax: 2.02 ± 0.15mN; pEC50: 4.32±0.06) and PZS 100nM (Emax 1.95 ± 0.21mN; pEC50: 3.65 ± 0.16) and in the presence of vehicle (Emax: 3.26 ± 0.64mN; pEC50: 4.79 ± 0.17). In the presence of Nm, 5-HT concentration-response curve showed the inhibition of contraction in 100nM (Emax: 1.98 ± 0.04mN; pEC50: 4.35± 0.02) and PZS 100nM (Emax: 2.26 ± 0.32mN; pEC50: 4.29), compared to vehicle (Emax: 2.52 ± 4.24mN; pEC50: 4.24 ± 0.10).

Limonin alleviates macro- and micro-vascular complications of metabolic syndrome in rats: A comparative study with azelnidipine

BackgroundHypertension is a serious component of metabolic syndrome (MetS). HypothesisThis research investigates the potential protective effect of limonin against MetS-associated hypertension in comparison with azelnidipine, a common calcium channel blocker. Study designMetS was induced in rats by 10% fructose in water and 3% salt in diet over a 16-week period. Limonin (50 mg/kg) and azelnidipine (5 mg/kg) were administered daily in the last four weeks MethodsNon-invasive blood pressure (BP) was recorded in conscious animals. Concentration-response curves for phenylephrine (PE) and acetylcholine (ACh) were analysed in thoracic aorta (macrovessels) and kidney microvessels. Blood glucose level, serum insulin level, advanced glycation end products (AGEs), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA) and transforming growth factor-β1 (TGF-β1) were determined. ResultsLimonin alleviated elevations in systolic and diastolic BP associated with MetS similar to levels associated with azelnidipine. Limonin prevented the MetS induced exaggerated macro- and micro-vascular contractility to PE and the impaired dilatation to ACh. However, in vitro incubation with limonin partially alleviated the deteriorated vascular reactivity of aorta isolated from MetS animals or AGEs injured aorta. Limonin did not have direct relaxant effect on the isolated vessel. On the other hand, limonin reduced the elevated serum levels of AGEs, TNF-α and MDA. Limonin suppressed the vascular fibrosis through reducing the elevated serum level of TGF-β1 and excessive aortic collagen deposition. Limonin decreased the elevated HOMA-IR in MetS animals. ConclusionLimonin offsets the hypertensive and vascular impairment associated with MetS via attenuation of inflammation and fibrosis. Its impact is comparable to that of azelnidipine.

Tributyltin chloride disrupts aortic vascular reactivity and increases reactive oxygen species production in female rats.

Organotin compounds, such as tributyltin (TBT), are environment contaminants that induce bioaccumulation and have potential toxic effects on marine species and mammals. TBT have been banned by the International Maritime Organization in 2003. However, the assessment of butyltin and metal contents in marine sediments has demonstrated high residual levels of TBT in some cases exceeding 7000ngSng-1. The acceptable daily intake (ADI) level for TBT established by the World Health Organization is 0.5μg/kg bw/day is based on genotoxicity, reproduction, teratogenicity, immunotoxicity, and mainly neurotoxicity. However, their effect on the cardiovascular system is not well understood. In this study, female rats were exposed to 0.5μg/kg/day of TBT for 15days with the goal of understanding the effect of TBT on vascular function. Female Wistar rats were treated daily by gavage and divided into control (n=10) and TBT (n=10) groups. The aortic rings were incubated with phenylephrine in both the presence and absence of endothelium. The phenylephrine concentration-response curves were generated by exposing endothelium-intact samples to NG-nitro-L-arginine methyl ester (L-NAME), apocynin, superoxide dismutase (SOD), catalase, tiron, and allopurinol. Acetylcholine (ACh) and sodium nitroprusside (SNP) were used to evaluate the relaxation response. Exposure to TBT reduced serum 17β-estradiol E2 levels and increased vascular reactivity. After incubation with L-NAME, the vascular reactivity to phenylephrine was significantly higher. Apocynin, SOD, catalase, and tiron decreased the vascular reactivity to phenylephrine to a significantly greater extent in TBT-treated rats than in the control rat. The relaxation induced by ACh and SNP was significantly reduced in TBT rats. Exposure to TBT induced aortic wall atrophy and increased superoxide anion production and collagen deposition. These results provide evidence that exposing rats to the current ADI for TBT (0.5μg/kg) for 15days induced vascular dysfunction due to oxidative stress and morphological damage and should be considered an important cardiovascular risk factor.

Elucidation of the profound antagonism of contractile action of phenylephrine in rat aorta effected by an atypical sympathomimetic decongestant.

Vasoconstrictive properties of sympathomimetic drugs are the basis of their widespread use as decongestants and possible source of adverse responses. Insufficiently substantiated practice of combining decongestants in some marketed preparations, such are those containing phenylephrine and lerimazoline, may affect the overall contractile activity, and thus their therapeutic utility. This study aimed to examine the interaction between lerimazoline and phenylephrine in isolated rat aortic rings, and also to assess the substrate of the obtained lerimazoline-induced attenuation of phenylephrine contraction. Namely, while lower concentrations of lerimazoline (10−6 M and especially 10−7 M) expectedly tended to potentiate the phenylephrine-induced contractions, lerimazoline in higher concentrations (10−4 M and above) unexpectedly and profoundly depleted the phenylephrine concentration-response curve. Suppression of NO with NO synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester (L-NAME; 10−4 M) or NO scavanger OHB12 (10−3 M), as well as non-specific inhibition of K+-channels with tetraethylammonium (TEA; 10−3 M), have reversed lerimazoline-induced relaxation of phenylephrine contractions, while cyclooxygenase inhibitor indomethacin (10−5 M) did not affect the interaction between two vasoconstrictors. At the receptor level, non-selective 5-HT receptor antagonist methiothepin reversed the attenuating effect of lerimazoline on phenylephrine contraction when applied at 3×10−7 and 10−6 M, but not at the highest concentration (10−4 M). Neither the 5-HT1D-receptor selective antagonist BRL 15572 (10−6 M) nor 5-HT7 receptor selective antagonist SB 269970 (10−6 M) affected the lerimazoline-induced attenuation of phenylephrine activity. The mechanism of lerimazoline-induced suppression of phenylephrine contractions may involve potentiation of activity of NO and K+-channels and activation of some methiothepin-sensitive receptors, possibly of the 5-HT2B subtype.

219 Stimulation of Sympathetic Nerves within Perivascular Adipose Tissue Causes the Release of Vasoactive Relaxing Factors

<h3>Introduction</h3> There is considerable evidence to implicate over-activity of the sympathetic nervous system during obesity and the subsequent development of metabolic syndrome and type 2 diabetes. It has been suggested that these conditions are dependent upon changes in small artery function. Healthy perivascular fat exerts an anti-contractile effect on circulation which is lost in obesity. Therefore it was decided to examine the effects of sympathetic nerve stimulation on perivascular adipose tissue (PVAT) function. The hypothesis to be tested was that an anti-contractile factor is released from PVAT as a result of sympathetic nerve stimulation. <h3>Methods</h3> The frequency-stimulation profiles (0.1–30 Hz) of murine mesenteric arteries (&lt;200 µM), +/-PVAT, were characterised using wire myography at various voltages (5–30 V). The accepted test for neural stimulation using 1 µM tetrodotoxin (TTX) was performed, and 1.46 µM 6-hydroxydopamine (6-OHDA) used to sympathetically denervate vessels +/-PVAT. Exogenous PVAT was incubated with TTX and 6-OHDA for 30 min before re-suspending in the bath. Specific adrenoceptor stimulation of PVAT function was investigated using noradrenaline, phenylephrine (both 1 × 10^–9–1 × 10^–5M), and CL-316,243 (10 µm). <h3>Results</h3> At 20&amp;30V PVAT elicited an anti-contractile effect (P &lt; 0.01); reproducible at 20V after a 15 min rest period. This effect is due to factors released from the PVAT and not due to a “barrier” effect, as application of exogenous PVAT elicits the same anti-contractile response (P &lt; 0.05). Sympathetic denervation of whole vessels with 6-OHDA abolished all significant contractile activity, confirming that EFS at 20V innervates sympathetic nerves (P &lt; 0.0001). PVAT incubated with TTX, demonstrated a reduced anti-contractile effect, confirming this effect to be neural and therefore physiologically relevant (P &lt; 0.05). Exogenous PVAT pre-incubated with 6-OHDA also induced a diminished PVAT anti-contractile effect, implicating the role of sympathetic nerves in PVAT (P &lt; 0.0001). In the noradrenaline but not phenylephrine concentration-response curve, PVAT elicits an anti-contractile response. The addition of β₃-adrenoceptor agonist CL-316,243 to phenylephrine-constricted +PVAT vessels induced a relaxation which replicated the PVAT anti-contractile effect seen in vessels stimulated with NA (P &lt; 0.05), indicating the role of β₃-adrenoceptors in PVAT. <h3>Conclusions</h3> These results clearly demonstrate sympathetic nerve stimulation can provoke the release of vasodilators from PVAT. The mechanism appears to be via activation of the adipocyte membrane located β3-adrenoreceptor. Further studies will be carried out in an obese model to understand how over-stimulation leads to a down-regulation and a loss of anti-contractility possibly via β3 receptor desensitisation.

Heme oxygenase-1 alleviates vascular complications associated with metabolic syndrome: Effect on endothelial dependent relaxation and NO production

Protective effect of Heme oxygenase-1 (HO-1) induction from hypertension was previously reported in a diabetic animal model. Here, the effect of HO-1 induction on vascular complications associated with metabolic syndrome (MetS) was investigated.MetS was induced in rats by fructose drinking for 12weeks while HO-1 was induced by hemin or curcumin administration in the last 6weeks. Then, aortic HO-1 protein expression was assessed, blood pressure (BP) was recorded and serum levels of glucose and insulin were measured. Concentration response curves for phenylephrine (PE), KCl, and acetylcholine (ACh) were obtained in thoracic aortic cross sections. Aortic reactive oxygen species (ROS) and nitric oxide (NO) generation were also studied.Both hemin and curcumin significantly inhibited the elevated systolic and diastolic BP seen in MetS animals. While not affected by MetS, HO-1 expression was significantly increased by hemin and curcumin treatment. HO-1 induction did not affect the exaggerated vasoconstriction response to KCl and PE. However, HO-1 induction prevented the impaired relaxation and NO generation in aorta isolated from MetS animals. In addition, the HO inhibitor, tin protoporphyrin, abolished the hemin protective effect on relaxation and NO generation. HO-1 induction prevented the elevated hyperinsulinemia associated with MetS. Furthermore, HO-1 induction inhibited ROS production in aorta isolated from MetS animals.In conclusion, Heme oxygenase-1 alleviates vascular complications associated in MetS through maintaining endothelial-dependent relaxation and NO generation in addition to improving insulin sensitivity.

Bupivacaine-induced Vasodilation Is Mediated by Decreased Calcium Sensitization in Isolated Endothelium-denuded Rat Aortas Precontracted with Phenylephrine.

BackgroundA toxic dose of bupivacaine produces vasodilation in isolated aortas. The goal of this in vitro study was to investigate the cellular mechanism associated with bupivacaine-induced vasodilation in isolated endotheliumdenuded rat aortas precontracted with phenylephrine.MethodsIsolated endothelium-denuded rat aortas were suspended for isometric tension recordings. The effects of nifedipine, verapamil, iberiotoxin, 4-aminopyridine, barium chloride, and glibenclamide on bupivacaine concentration-response curves were assessed in endothelium-denuded aortas precontracted with phenylephrine. The effect of phenylephrine and KCl used for precontraction on bupivacaine-induced concentration-response curves was assessed. The effects of verapamil on phenylephrine concentration-response curves were assessed. The effects of bupivacaine on the intracellular calcium concentration ([Ca2+]i) and tension in aortas precontracted with phenylephrine were measured simultaneously with the acetoxymethyl ester of a fura-2-loaded aortic strip.ResultsPretreatment with potassium channel inhibitors had no effect on bupivacaine-induced relaxation in the endothelium-denuded aortas precontracted with phenylephrine, whereas verapamil or nifedipine attenuated bupivacaine-induced relaxation. The magnitude of the bupivacaine-induced relaxation was enhanced in the 100 mM KCl-induced precontracted aortas compared with the phenylephrine-induced precontracted aortas. Verapamil attenuated the phenylephrine-induced contraction. The magnitude of the bupivacaine-induced relaxation was higher than that of the bupivacaine-induced [Ca2+]i decrease in the aortas precontracted with phenylephrine.ConclusionsTaken together, these results suggest that toxic-dose bupivacaine-induced vasodilation appears to be mediated by decreased calcium sensitization in endothelium-denuded aortas precontracted with phenylephrine. In addition, potassium channel inhibitors had no effect on bupivacaine-induced relaxation. Toxic-dose bupivacaine- induced vasodilation may be partially associated with the inhibitory effect of voltage-operated calcium channels.

Perivascular adipose tissue inhibits endothelial function of rat aortas via caveolin-1.

Perivascular adipose tissue (PVAT)-derived factors have been proposed to play an important role in the pathogenesis of atherosclerosis. Caveolin-1 (Cav-1), occupying the calcium/calmodulin binding site of endothelial NO synthase (eNOS) and then inhibiting nitric oxide (NO) production, is also involved in the development of atherosclerosis. Thus, we investigated whether PVAT regulated vascular tone via Cav-1 and/or endothelial NO pathways. Isometric tension studies were carried out in isolated thoracic aortas from Wistar rats in the presence and absence of PVAT. Concentration-response curves of phenylephrine, acetylcholine, and sodium nitroprusside were illustrated to examine the vascular reactivity and endothelial function. The protein expressions of eNOS and Cav-1 were also examined in aortic homogenates. Our results demonstrated that PVAT significantly enhanced vasoconstriction and inhibited vasodilatation via endothelium-dependent mechanism. The aortic NO production was diminished after PVAT treatment, whereas protein expression and activity of eNOS were not significantly affected. In addition, Cav-1 protein expression was significantly increased in aortas with PVAT transfer. Furthermore, a caveolae depleter methyl-β-cyclodextrin abolished the effect of PVAT on the enhancement of vasoconstriction, and reversed the impairment of aortic NO production. In conclusion, unknown factor(s) released from PVAT may inhibit endothelial NO production and induce vasocontraction via an increase of Cav-1 protein expression.

Chrysin and luteolin alleviate vascular complications associated with insulin resistance mainly through PPAR-γ activation.

Chrysin and luteolin are two flavonoids with Peroxisome proliferators-activated receptor γ (PPAR-γ) stimulating activity. Here, we investigated the protective effect of chrysin and luteolin from vascular complications associated with insulin resistance (IR). IR was induced in rats by drinking fructose for 12 weeks while chrysin and luteolin were given for 6 weeks with or without PPAR-γ antagonist, bisphenol A diglycidyl ether (BADGE). Then, blood pressure (BP) was recorded and serum levels of glucose, insulin, advanced glycation end products (AGEs) and lipids were measured. Concentration response curves for phenylephrine (PE), KCl, and acetylcholine (ACh) were obtained in thoracic aorta rings. Aortic reactive oxygen species (ROS) and nitric oxide (NO) generation were also studied. Chrysin and luteolin significantly alleviated systolic BP elevations caused by IR, while the co-administration of BADGE prevented chrysin alleviation. Although, neither chrysin nor luteolin affected ACh impaired vasodilatation, they both alleviated exaggerated vasoconstrictions to PE and KCl in IR animals. In addition, incubation of the aorta from IR animals with chrysin or luteolin prevented exaggerated vasoconstrictions to PE and KCl. On the other hand, co-administration of BADGE or co-incubation with GW9662, the selective PPAR-γ antagonist, prevented chrysin alleviation. Both chrysin and luteolin inhibited the developed hyperinsulinemia and increases in serum AGEs, lipids while, BADGE reduced the effect of chrysin on hyperinsulinemia and dyslipidemia. Chrysin and luteolin markedly inhibited elevated NO and ROS in IR aortae while BADGE did not change their effect on NO and ROS. In conclusion, chrysin and luteolin alleviate vascular complications associated with IR mainly through PPAR-γ dependent pathways.

Quercetin Protects against Diabetes-Induced Exaggerated Vasoconstriction in Rats: Effect on Low Grade Inflammation

Vascular complications are the leading cause of morbidity and mortality in patients with diabetes. Quercetin is an important flavonoid with antioxidant and anti-inflammatory activity. Here, the effect of quercetin on diabetes-induced exaggerated vasoconstriction in insulin deficient and insulin resistant rat models was investigated. Insulin deficiency was induced by streptozotocin while, insulin resistance by fructose. Rats were left 8 weeks or 12 weeks after STZ or fructose administration respectively. Quercetin was daily administered in the last 6 weeks. Then, tail blood pressure (BP) was recorded in conscious animals; concentration-response curves for phenylephrine (PE) and KCl were studied in thoracic aorta rings. Non-fasting blood glucose level, serum insulin level, insulin resistance index, serum tumour necrosis factor-α (TNF-α) and serum C-reactive protein (CRP) were determined. Nuclear transcription factor-κB (NF-κB) was assessed by immunofluorescence technique. Histopathological examination was also performed. The results showed that quercetin protected against diabetes-induced exaggerated vasoconstriction and reduced the elevated blood pressure. In addition, quercetin inhibited diabetes associated adventitial leukocyte infiltration, endothelial pyknosis and increased collagen deposition. These effects were accompanied with reduction in serum level of both TNF-α and CRP and inhibition of aortic NF-κB by quercetin in both models of diabetes. On the other hand, quercetin did not affect glucose level in any of the used diabetic models. This suggests that the protective effect of quercetin is mediated by its anti-inflammatory effect rather than its metabolic effects. In summary, quercetin is potential candidate to prevent diabetic vascular complications in both insulin deficiency and resistance via its inhibitory effect on inflammatory pathways especially NF-κB signaling.

Arginase inhibition alleviates hypertension in the metabolic syndrome

We have previously shown that arginase inhibition alleviates hypertension associated with in a diabetic animal model. Here, we investigated the protective effect of arginase inhibition on hypertension in metabolic syndrome. Metabolic syndrome was induced in rats by administration of fructose (10% in drinking water) for 12 weeks to induce vascular dysfunction. Three arginase inhibitors (citrulline, norvaline and ornithine) were administered daily in the last 6 weeks of study before and tail BP was recorded in conscious animals. Concentration response curves for phenylephrine (PE), KCl and ACh in addition to ACh-induced NO generation were obtained in thoracic aorta rings. Serum glucose, insulin, uric acid and lipid profile were determined as well as reactive oxygen species (ROS) and arginase activity. Arginase activity was elevated in metabolic syndrome while significantly inhibited by citrulline, norvaline or ornithine treatment. Metabolic syndrome was associated with elevations in systolic and diastolic BP, while arginase inhibition significantly reduced elevations in diastolic and systolic BP. Metabolic syndrome increased vasoconstriction responses of aorta to PE and KCl and decreased vasorelaxation to ACh, while arginase inhibition completely prevented impaired responses to ACh. In addition, arginase inhibition prevented impaired NO generation and exaggerated ROS formation in metabolic syndrome. Furthermore, arginase inhibition significantly reduced hyperinsulinaemia and hypertriglyceridaemia without affecting hyperuricaemia or hypercholesterolaemia associated with metabolic syndrome. Arginase inhibition alleviates hypertension in metabolic syndrome directly through endothelial-dependent relaxation/NO signalling protection and indirectly through inhibition of insulin resistance and hypertriglyceridaemia.

Small arteries of gestational rats are less sensitive to relaxation induced by inhibition of rho A‐associated kinase (ROK).

Inhibition of ROK has been proposed as a method to arrest uterine contractions during preterm labor. Because of concern about vascular side effects, we studied the effects of the ROK inhibitor g‐H1152 (g‐H) on relaxation of mesenteric and uterine arteries.Arteries from non‐pregnant (n=5) and pregnant (gestation day17–21; n=6) rats were studied using wire myography. Concentration response curves (CRC) to calculate IC50 for g‐H were obtained following phenylephrine (PE) or endothelin‐1 (ET‐1) induced constriction. Using vessels from pregnant rats, CRC for PE were performed before and after a 20‐min incubation with g‐H at 300 nM, 1 and 3 μM.Mesenteric arteries were more sensitive than uterine arteries to g‐H‐induced relaxation in both non‐pregnant and pregnant rats. Both arteries were significantly less sensitive to g‐H‐induced relaxation during pregnancy. Sensitivity to PE was not changed with any dose of g‐H. However, using 2‐factor ANOVA, the maximal PE‐induced tension was significantly reduced by the two highest concentrations of g‐H in both vessels from pregnant rats but to a greater extent in mesenteric vessels (34±5.6%, 18±4.1%) compared to uterine (14±4%, 7±2.7%).The ROK pathway plays a significant role in agonist‐induced constriction of mesenteric and uterine vessels, though less so during gestation. If ROK inhibitors are used during pregnancy in vivo, maternal blood pressure and uterine blood flow will be assessed.