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Background and Purpose Experimental Approach Key Results Conclusions and Implications In male rats, the serotonergic system modulates sympathetic outflow at vascular levels, causing sympatho‐inhibition and sympatho‐excitation, mainly via 5‐HT1D/1A and 5‐HT3 receptors, respectively. However, sex influence on vascular serotonergic regulation has not yet been elucidated. This study aimed to analyse the 5‐HT sympatho‐modulatory role in female rats, characterising the 5‐HT receptors involved.Female Wistar (14‐ to 16‐week‐old) rats were prepared for sympathetic stimulation. Mean blood pressure (MBP) and heart rate (HR) were continuously measured. Vasopressor responses were obtained by electrical stimulation of the sympathetic outflow (0.1–5 Hz) or i.v. noradrenaline (0.01–0.5 μg·kg−1). 5‐HT‐related drug effects on adrenergic system were determined. Age‐matched male rats were used as control.Basal MBP in females was lower than in male rats, whereas electrical‐induced increases in MBP were similar. In females, 5‐HT exerted a dose‐dependent inhibition on the sympathetic‐evoked vasoconstrictions, that was reproduced by some agonists; 5‐CT (5‐HT1/5/7) and L‐694,247 (5‐HT1D), whereas the selective 5‐HT2A/2B/2C (α‐methyl‐5‐HT) and 5‐HT3 agonist (1‐PBG) increased the electrically‐produced vasopressor responses. None of the other drugs tested (targeting 5‐HT1A/1B/1F, 5‐HT2B/2C, 5‐HT4, 5‐HT5A or 5‐HT7) modified these vasoconstrictions. Only 1‐PBG (5‐HT3) modified the vasoconstrictions induced by exogenous noradrenaline.In female rats, vascular serotonergic sympatholytic effects are due to prejunctional 5‐HT1D receptor activation, whereas pre and/or postjunctional 5‐HT3 and prejunctional 5‐HT2A receptor activation is involved in the potentiating effect of vascular sympathetic neurotransmission. These findings may open novel sex‐differential therapeutic strategies for treating cardiovascular conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: Raised serum concentrations of the sympathetic co‐transmitter neuropeptide Y (NPY) are linked to cardiovascular diseases. However, the signalling mechanism for vascular smooth muscle (VSM) constriction to NPY is poorly understood. Therefore, the present study investigated the mechanisms of NPY‐induced vasoconstriction in rat small mesenteric (RMA) and coronary (RCA) arteries. Experimental Approach: Third‐order mesenteric or intra‐septal arteries from male Wistar rats were assessed in wire myographs for isometric tension, VSM membrane potential and VSM intracellular Ca2+ events. Key Results: NPY stimulated concentration‐dependent vasoconstriction in both RMA and RCA, which was augmented by blocking NO synthase or endothelial denudation in RMA. NPY‐mediated vasoconstriction was blocked by the selective Y1 receptor antagonist BIBO 3304 and Y1 receptor protein expression was detected in both the VSM and endothelial cells in RMA and RCA. The selective Gβγ subunit inhibitor gallein and the PLC inhibitor U‐73122 attenuated NPY‐induced vasoconstriction. Signalling via the Gβγ–PLC pathway stimulated VSM Ca2+ waves and whole‐field synchronised Ca2+ flashes in RMA and increased the frequency of Ca2+ flashes in myogenically active RCA. Furthermore, in RMA, the Gβγ pathway linked NPY to VSM depolarization and generation of action potential‐like spikes associated with intense vasoconstriction. This depolarization activated L‐type voltage‐gated Ca2+ channels, as nifedipine abolished NPY‐mediated vasoconstriction. Conclusions and Implications: These data suggest that the Gβγ subunit, which dissociates upon Y1 receptor activation, initiates VSM membrane depolarization and Ca2+ mobilisation to cause vasoconstriction. This model may help explain the development of microvascular vasospasm during raised sympathetic nerve activity. [ABSTRACT FROM AUTHOR]

Background and Purpose: Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9β1, and SVEP1, a ligand for integrin α9β1, associate with elevated blood pressure; however, neither SVEP1 nor integrin α9β1 has reported roles in vasoregulation. We determined whether SVEP1 and integrin α9β1 can regulate VSMC contraction.Experimental Approach: SVEP1 and integrin binding were confirmed by immunoprecipitation and cell binding assays. Human induced pluripotent stem cell-derived VSMCs were used in in vitro [Ca2+ ]i studies, and aortas from a Svep1+/- knockout mouse model were used in wire myography to measure vessel contraction.Key Results: We confirmed the ligation of SVEP1 to integrin α9β1 and additionally found SVEP1 to directly bind to integrin α4β1. Inhibition of SVEP1, integrin α4β1 or α9β1 significantly enhanced [Ca2+ ]i levels in isolated VSMCs to Gαq/11 -vasoconstrictors. This response was confirmed in whole vessels where a greater contraction to U46619 was seen in vessels from Svep1+/- mice compared to littermate controls or when integrin α4β1 or α9β1 was inhibited. Inhibition studies suggested that this effect was mediated via VGCCs, PKC and Rho A/Rho kinase dependent mechanisms.Conclusions and Implications: Our studies reveal a novel role for SVEP1 and the integrins α4β1 and α9β1 in reducing VSMC contractility. This could provide an explanation for the genetic associations with blood pressure risk at the SVEP1 and ITGA9 loci. [ABSTRACT FROM AUTHOR]

Background and Purpose: l‐cysteine or hydrogen sulfide (H2S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 μM followed by vasodilatation at 30–100 μM. Here, we have investigated the signalling involved in the H2S‐induced contraction. Experimental Approach Vascular response to NaHS or l‐cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ‐lyase (CSE−/−), soluble guanylyl cyclase (sGCα1−/−) and endothelial nitric oxide synthase (eNOS−/−) knock‐out mice. The cAMP, cGMP and inosine 3′,5′‐cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated. Key Results: CSE‐derived H2S‐induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H2S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell‐permeable analogue of cIMP elicits concentration‐dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE−/− mice, confirms that H2S‐induced contraction involves cIMP. Conclusion and Implications: The endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE‐derived H2S that is mediated by cIMP. [ABSTRACT FROM AUTHOR]

Background and Purpose: Reduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso‐occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP‐amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα‐induced inflammation in wild‐type C57BL/6 mice and in 'humanised' mouse models of SCD. Experimental Approach Effects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα‐induced and systemic inflammation associated with SCD. Key Results: Acute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte‐endothelial cell interactions in TNFα‐challenged mice. Co‐treatment with hydroxyurea, an FDA‐approved SCD therapeutic agent, further augmented the anti‐inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα‐induced vaso‐occlusive crisis, a single dose of olinciguat attenuated leukocyte‐endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle‐treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat‐ than in vehicle‐treated mice. In addition, kidney mass, water consumption, 24‐h urine excretion, plasma levels of cystatin C and urinary excretion of N‐acetyl‐β‐d‐glucosaminidase and neutrophil gelatinase‐associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle‐treated mice. Conclusion and Implications: Our results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso‐occlusion and kidney injury in mouse models of SCD and systemic inflammation. [ABSTRACT FROM AUTHOR]

Background and Purpose: Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti‐hypertrophic, anti‐fibrotic and anti‐proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia‐induced pulmonary hypertension (HPH). Experimental Approach Patch‐clamp experiments were conducted to examine the activity of voltage‐dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2) for 4 weeks were used as model of HPH for in vivo experiments. Key Results: Halofuginone increased voltage‐gated K+ (Kv) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03–1 μM) inhibited receptor‐operated Ca2+ entry in HEK cells transfected with calcium‐sensing receptor gene and attenuated store‐operated Ca2+ entry in PASMCs. Acute (3–5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia‐induced pulmonary vasoconstriction dose‐dependently (0.1–10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg−1, for 2 weeks) partly reversed established PH in mice. Conclusion and Implications: Halofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor‐operated and store‐operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation–contraction coupling and anti‐proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway. [ABSTRACT FROM AUTHOR]

Background and Purpose: The TRPV cation channels have emerged as important regulators of vascular tone. TRPV1 channels and endothelin-1 are independently associated with the pathophysiology of coronary vasospasm, but the relationship between their vasomotor functions remains unclear. We characterized the vasomotor function of TRPV1 channels in human arterioles and investigated regulation of their vasomotor function by endothelin-1.Experimental Approach: Human arterioles (mainly from adipose tissue) were threaded on two metal wires, equilibrated in a physiological buffer at 37°C and exposed to increasing concentrations of capsaicin, with or without SB366791 (TRPV1-selective inhibitor) or GF109203X (PKC-selective inhibitor). Some arterioles were pre-constricted with endothelin-1 or phenylephrine or high potassium buffer. TRPV1 mRNA and protein expression in human arteries were also assessed.Key Results: TRPV1 transcripts and proteins were detected in human resistance arteries. Capsaicin (1 μM) induced concentration-dependent constriction of endothelium-intact and endothelium-denuded human adipose arterioles (HAA), which was significantly inhibited by SB366791. Pre-constriction of HAA with endothelin-1, but not high potassium buffer or phenylephrine, significantly potentiated capsaicin (0.1 μM)-induced constriction. GF109203X significantly inhibited potentiation of capsaicin-induced constriction by endothelin-1.Conclusion and Implications: TRPV1 channels are expressed in the human vasculature and affect vascular tone of human arterioles on activation. Their vasomotor function is modulated by endothelin-1, mediated in part by PKC. These findings reveal a novel interplay between endothelin-1 signalling and TRPV1 channels in human VSMC, adding to our understanding of the ion channel mechanisms that regulate human arteriolar tone and may also contribute to the pathophysiology of coronary vasospasm. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hydrogen sulfide (H2 S)-releasing agents are viewed as potential antihypertensive drugs. Recently, natural isothiocyanates emerged as original H2 S-donor agents. Among them, erucin, present in some edible cruciferous plants, shows suitable H2 S-releasing properties and features of "druggability." The aim of this work was to investigate the erucin-mediated release of H2 S inside vascular cells, its vasorelaxing effects, and activity on BP of normo and hypertensive animals.Experimental Approach: Intracellular H2 S-release and the hyperpolarizing effect of erucin were tested using fluorescent dye, in human aortic smooth muscle cells (HASMCs). Its direct vasorelaxing effect and ability to inhibit noradrenaline-induced vasoconstriction were evaluated on endothelium-intact or -denuded rat aortic rings. Its vasodilator properties were tested in coronary arteries using Langendorff-perfused rat hearts. Finally, erucin's antihypertensive activity was evaluated in vivo in normotensive and spontaneously hypertensive rats (SHRs) by recording systolic BP using the tail-cuff method.Key Results: Erucin induced the release of H2 S inside HASMCs. Moreover, erucin hyperpolarized the membrane of HASMCs membrane in a concentration-dependent manner. It induced vasodilatation of rat aortic rings, in endothelium-denuded vessels. This effect was further improved by the presence of endothelial NO. When pre-incubated with rat aortic rings, erucin induced concentration-dependent inhibition of noradrenaline-induced vasoconstriction. Erucin did not affect basal coronary flow but restored the flow to normal in pre-contracted coronary vessels. Finally, in vivo, erucin decreased systolic BP in SHRs by about 25%, and restored the BP to values observed in normotensive rats.Conclusions and Implications: Erucin is an H2 S donor endowed with vasorelaxing and antihypertensive effects.Linked Articles: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc. [ABSTRACT FROM AUTHOR]

Background and Purpose: Adenosine is a local mediator that regulates physiological and pathological processes via activation of four GPCRs (A1 , A2A , A2B , and A3 ). We have investigated the effect of two A1 -receptor-selective agonists and the novel A1 -receptor bitopic ligand VCP746 on the rat cardiovascular system.Experimental Approach: The regional haemodynamic responses of these agonist was investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal, mesenteric arteries and the descending abdominal aorta and the jugular vein and caudal artery catheterized. Cardiovascular responses were measured following intravenous infusion (3 min each dose) of CCPA (120, 400, and 1,200 ng·kg-1 ·min-1 ), capadenoson or adenosine (30, 100, and 300 μg·kg-1 ·min-1 ), or VCP746 (6, 20, and 60 μg·kg-1 ·min-1 ) following pre-dosing with DPCPX (0.1 mg·kg-1 , i.v.) or vehicle.Key Results: CCPA produced a significant A1 -receptor-mediated decrease in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A1 -receptor-mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A1 -receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A2A - and A2B -receptors.Conclusions and Implications: These results suggest VCP746 mediates its cardiovascular effects via activation of A2 rather than A1 adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A1 allosteric ligand moieties. [ABSTRACT FROM AUTHOR]

Background and Purpose: Endothelium-derived vasoconstriction is a hallmark of vascular dysfunction in hypertension. In some cases, an overproduction of endothelium-derived prostacyclin (PGI2 ) can cause contraction rather than relaxation. Relaxin is well known for its vasoprotective actions, but the possibility that this peptide could also reverse endothelium-derived vasoconstriction has never been investigated. We tested the hypothesis that short-term relaxin treatment mitigates endothelium-derived vasoconstriction in spontaneously hypertensive rats (SHR).Experimental Approach: Male Wistar Kyoto rats (WKY) and SHR were subcutaneously infused with either vehicle (20 mmol·L-1 sodium acetate) or relaxin (13.3 μg·kg-1 ·hr-1 ) using osmotic minipumps for 3 days. Vascular reactivity to the endothelium-dependent agonist ACh was assessed in vitro by wire myography. Quantitative PCR and LC-MS were used to identify changes in gene expression of prostanoid pathways and PG production, respectively.Key Results: Relaxin treatment ameliorated hypertension-induced endothelial dysfunction by increasing NO-dependent relaxation and reducing endothelium-dependent contraction. Notably, short-term relaxin treatment up-regulated mesenteric PGI2 receptor (IP) expression, permitting PGI2 -IP-mediated vasorelaxation. In the aorta, reversal of contraction was accompanied by suppression of the hypertension-induced increase in prostanoid-producing enzymes and reduction in PGI2 -evoked contractions.Conclusions and Implications: Relaxin has region-dependent vasoprotective actions in hypertension. Specifically, relaxin has distinct effects on endothelium-derived contracting factors and their associated vasoconstrictor pathways in mesenteric arteries and the aorta. Taken together, these observations reveal the potential of relaxin as a new therapeutic agent for vascular disorders that are associated with endothelium-derived vasoconstriction including hypertension. [ABSTRACT FROM AUTHOR]

Background and Purpose: Angiotensin II (AngII) and NO regulate the cerebral circulation. AngII AT1 receptors exert ligand‐dependent and ligand‐independent (myogenic tone [MT]) vasoconstriction of cerebral vessels. NO induces post‐translational modifications of proteins such as S‐nitrosation (redox modification of cysteine residues). In cultured cells, S‐nitrosation decreases AngII's affinity for the AT1 receptor. The present work evaluated the functional consequences of S‐nitrosation on both AngII‐dependent and AngII‐independent cerebrovascular responses. Experimental Approach S‐Nitrosation was induced in rat isolated middle cerebral arteries by pretreatment with the NO donors, S‐nitrosoglutathione (GSNO) or sodium nitroprusside (SNP). Agonist‐dependent activation of AT1 receptors was evaluated by obtaining concentration–response curves to AngII. Ligand‐independent activation of AT1 receptors was evaluated by calculating MT (active vs. passive diameter) at pressures ranging from 20 to 200 mmHg in the presence or not of a selective AT1 receptor inverse agonist. Key Results: GSNO or SNP completely abolished the AngII‐dependent AT1 receptor‐mediated vasoconstriction of cerebral arteries. GSNO had no impact on responses to other vasoconstrictors sharing (phenylephrine, U46619) or not (5‐HT) the same signalling pathway. MT was reduced by GSNO, and the addition of losartan did not further decrease MT, suggesting that GSNO blocks AT1 receptor‐dependent MT. Ascorbate (which reduces S‐nitrosated compounds) restored the response to AngII but not the soluble GC inhibitor ODQ, suggesting that these effects are mediated by S‐nitrosation rather than by S‐nitrosylation. Conclusions and Implications: In rat middle cerebral arteries, GSNO pretreatment specifically affects the AT1 receptor and reduces both AngII‐dependent and AngII‐independent activation, most likely through AT1 receptor S‐nitrosation. [ABSTRACT FROM AUTHOR]

Background and Purpose: Development of combination therapies has received significant interest in recent years. Previously, a two-receptor one-transducer (2R-1T) model was proposed to characterize drug interactions with two receptors that lead to the same phenotypic response through a common transducer pathway. We applied, for the first time, the 2R-1T model to characterize the interaction of noradrenaline and arginine-vasopressin on vasoconstriction and performed inter-species scaling to humans using this mechanism-based model.Experimental Approach: Contractile data were obtained from in vitro rat small mesenteric arteries after exposure to single or combined challenges of noradrenaline and arginine-vasopressin with or without pretreatment with the irreversible α-adrenoceptor antagonist, phenoxybenzamine. Data were analysed using the 2R-1T model to characterize the observed exposure-response relationships and drug-drug interaction. The model was then scaled to humans by accounting for differences in receptor density.Key Results: With receptor affinities set to published values, the 2R-1T model satisfactorily characterized the interaction between noradrenaline and arginine-vasopressin in rat small mesenteric arteries (relative standard error ≤20%), as well as the effect of phenoxybenzamine. Furthermore, after scaling the model to human vascular tissue, the model also adequately predicted the interaction between both agents on human renal arteries.Conclusions and Implications: The 2R-1T model can be of relevance to quantitatively characterize the interaction between two drugs that interact via different receptors and a common transducer pathway. Its mechanistic properties are valuable for scaling the model across species. This approach is therefore of significant value to rationally optimize novel combination treatments. [ABSTRACT FROM AUTHOR]

Background and Purpose: Kv 7.4 and Kv 7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (KV ) channels, with little inhibitory effect on Kv 7 family members at mmol concentrations. However, the effect of 4-AP on Kv 7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on Kv 7.4 and Kv 7.5 channels and characterize the effect of 4-AP on rat resistance arteries.Experimental Approach: Voltage clamp experiments were performed on Xenopus laevis oocytes injected with cRNA encoding KCNQ4 or KCNQ5, HEK cells expressing Kv 7.4 channels and on rat, freshly isolated mesenteric artery smooth muscle cells. The effect of 4-AP on tension, membrane potential, intracellular calcium and pH was assessed in rat mesenteric artery segments.Key Results: 4-AP increased the Kv 7.4-mediated current in oocytes and HEK cells but did not affect Kv 7.5 current. 4-AP also enhanced native mesenteric artery myocyte K+ current at sub-mmol concentrations. When applied to NA-preconstricted mesenteric artery segments, 4-AP hyperpolarized the membrane, decreased [Ca2+ ]i and caused concentration-dependent relaxations that were independent of 4-AP-mediated changes in intracellular pH. Application of the Kv 7 channel blocker XE991 and BKCa channel blocker iberiotoxin attenuated 4-AP-mediated relaxation. 4-AP also inhibited the NA-mediated signal transduction to elicit a relaxation.Conclusions and Implications: These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of Kv 7.4 and BKCa channels and attenuating NA-mediated signalling. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ethanol at concentrations obtained in the circulation during moderate-heavy episodic drinking (30-60 mM) causes cerebral artery constriction in several species, including humans. In rodents, ethanol-induced cerebral artery constriction results from ethanol inhibition of large conductance voltage/Ca2+i -gated K+ (BK) channels in cerebral artery myocytes. Moreover, the smooth muscle-abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral artery myocyte BK channels under physiological Ca2+i and voltages and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol-induced inhibition of cerebral artery myocyte BK channels and eventual arterial constriction.Experimental Approach: We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 K/O mouse arteries and artery in vitro pressurization.Key Results: Ethanol inhibition of BK current was facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 transmembrane (TM) domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wild-type β1. Moreover, β1 TM2 itself but not other β regions were necessary for ethanol-induced cerebral artery constriction.Conclusions and Implications: BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral artery constriction at physiological Ca2+ and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral artery constriction and associated cerebrovascular conditions. [ABSTRACT FROM AUTHOR]

Under physiological conditions, perivascular adipose tissue (PVAT) attenuates agonist-induced vasoconstriction by releasing vasoactive molecules including hydrogen peroxide, angiotensin 1-7, adiponectin, methyl palmitate, hydrogen sulfide, NO and leptin. This anticontractile effect of PVAT is lost under conditions of obesity. The central mechanism underlying this PVAT dysfunction in obesity is likely to be an 'obesity triad' (consisting of PVAT hypoxia, inflammation and oxidative stress) that leads to the impairment of PVAT-derived vasoregulators. The production of hydrogen sulfide, NO and adiponectin by PVAT is reduced in obesity, whereas the vasodilator response to leptin is impaired (vascular leptin resistance). Strikingly, the vasodilator response to acetylcholine is reduced only in PVAT-containing, but not in PVAT-free thoracic aorta isolated from diet-induced obese mice, indicating a unique role for PVAT in obesity-induced vascular dysfunction. Furthermore, PVAT dysfunction has also been observed in small arteries isolated from the gluteal/visceral fat biopsy samples of obese individuals. Therefore, PVAT may represent a new therapeutic target for vascular complications in obesity. A number of approaches are currently being tested under experimental conditions. Potential therapeutic strategies improving PVAT function include body weight reduction, enhancing PVAT hydrogen sulfide release (e.g. rosiglitazone, atorvastatin and cannabinoid CB1 receptor agonists) and NO production (e.g. arginase inhibitors), inhibition of the renin-angiotensin-aldosterone system, inhibition of inflammation with melatonin or cytokine antagonists, activators of AMP-activated kinase (e.g. metformin, resveratrol and diosgenin) and adiponectin releasers or expression enhancers.Linked Articles: This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc. [ABSTRACT FROM AUTHOR]

Background and Purpose: Perivascular adipose tissue (PVAT) surrounds most blood vessels and secretes numerous active substances, including adiponectin, which produce a net anticontractile effect in healthy individuals. AMPK is a key mediator of cellular energy balance and may mediate the vascular effects of adiponectin. In this study, we investigated the role of AMPK within PVAT in mediating the anticontractile effect of PVAT.Experimental Approach: Endothelium-denuded aortic rings from wild-type (WT; Sv129) and α1 AMPK knockout (KO) mice were mounted on a wire myograph. Dose-response curves to the AMPK-independent vasodilator cromakalim were studied in vessels with and without PVAT, and effect of pre-incubation with conditioned media and adiponectin on relaxation was also studied. The effect of AMPKα1 KO on the secretory profile of PVAT was assessed by elisa.Key Results: Thoracic aortic PVAT from KO mice was morphologically indistinct from that of WT and primarily composed of brown adipose tissue. PVAT augmented relaxation to cromakalim in WT but not KO aortic rings. Addition of WT PVAT augmented relaxation in KO aortic rings but KO PVAT had no effect in WT rings. PVAT from KO mice secreted significantly less adiponectin and addition of adiponectin to either KO or WT aortic rings without PVAT augmented relaxation to cromakalim. An adiponectin blocking peptide significantly attenuated relaxation in WT rings with PVAT but not in KO rings.Conclusions and Implications: AMPKα1 has a critical role in maintaining the anticontractile actions of PVAT; an effect independent of the endothelium but likely mediated through altered adiponectin secretion or sensitivity.Linked Articles: This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc. [ABSTRACT FROM AUTHOR]

Background and Purpose: As there is sexual dimorphism in the regulation of vascular tone, the aim of this present study was to determine whether there are sex differences in perivascular adipose tissue (PVAT)-mediated regulation of the porcine coronary artery (PCA) tone.Experimental Approach: Isometric tension recording system was used to record changes in tone in PCAs. Western blot analysis was performed to examine the expression of adiponectin in PVAT and adiponectin receptors and adiponectin binding protein (APPL1) in PCA. The level of adiponectin released from PVAT was measured using elisa.Key Results: In the presence of adherent PVAT, contractions to the thromboxane mimetic U46619 and endothelin-1 were significantly reduced in PCAs from females, but not males. In PCAs pre-contracted with U46619, re-addition of PVAT caused relaxation in PCAs from females, but not males. This relaxant response in females was attenuated by combined inhibition of NOS (with L-NAME) and COX (with indomethacin). Pre-incubation with an anti-adiponectin antibody abolished the relaxant effects of PVAT. The adiponectin receptor agonist (adipoRon) produced a greater relaxation in PCAs from females compared with males. However, there was no difference in either the expression or release of adiponectin from PVAT between sexes. Similarly, there was no difference in the expression of adiponectin receptors or the adiponectin receptor adaptor protein APPL1 in PCAs.Conclusion and Implications: These findings demonstrate a clear sex difference in the regulation of coronary arterial tone in response to adiponectin receptor stimulation, which may underlie the anticontractile effects of PVAT in females. [ABSTRACT FROM AUTHOR]

Background and Purpose: Oxidative stress plays a key role in the vascular and metabolic abnormalities associated with obesity. Herein, we assessed whether obesity can increase coronary vasoconstriction induced by hydrogen peroxide (H2 O2 ) and the signalling pathways involving COX-2 and superoxide (O2.- ) generation.Experimental Approach: Contractile responses to H2 O2 and O2.- generation were measured in coronary arteries from genetically obese Zucker rats (OZR) and compared to lean Zucker rats (LZR).Key Results: Both basal and H2 O2 -stimulated O2.- production were enhanced in coronary arteries from OZR, but H2 O2 -induced vasoconstriction was unchanged. The selective COX-2 inhibitor NS398 significantly reduced H2 O2 -induced contractions in endothelium-denuded arteries from LZR and OZR, but only in endothelium-intact arteries from LZR. PGI2 (IP) receptor antagonism modestly reduced the vasoconstrictor action of H2 O2 while antagonism of the PGE2 receptor 4 (EP4 ) enhanced H2 O2 contractions in arteries from OZR but not LZR. Basal release of COX-2-derived PGE2 was higher in coronary arteries from OZR where the selective agonist of EP4 receptors TCS 2519 evoked potent relaxations. COX-2 was up-regulated after acute exposure to H2 O2 in coronary endothelium and vascular smooth muscle (VSM) and inhibition of COX-2 markedly reduced H2 O2 -elicited O2.- generation in coronary arteries and myocardium. Expression of Nox subunits in VSM and NADPH-stimulated O2.- generation was enhanced and contributed to H2 O2 vasoconstriction in arteries from obese rats.Conclusion and Implications: COX-2 contributes to cardiac oxidative stress and to the endothelium-independent O2.- -mediated coronary vasoconstriction induced by H2 O2 in obesity, which is offset by the release of COX-2-derived endothelial PGE2 acting on EP4 vasodilator receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose: Nuciferine, a constituent of lotus leaf, is an aromatic ring-containing alkaloid, with antioxidative properties. We hypothesize nuciferine might affect vascular reactivity. This study aimed at determining the effects of nuciferine on vasomotor tone and the underlying mechanismExperimental Approach: Nuciferine-induced relaxations in rings of rat main mesenteric arteries were measured by wire myographs. Endothelial NOS (eNOS) was determined by immunoblotting. Intracellular NO production in HUVECs and Ca(2+) level in both HUVECs and vascular smooth muscle cells (VSMCs) from rat mesenteric arteries were assessed by fluorescence imaging.Key Results: Nuciferine induced relaxations in arterial segments pre-contracted by KCl or phenylephrine. Nuciferine-elicited arterial relaxations were reduced by removal of endothelium or by pretreatment with the eNOS inhibitor L-NAME or the NO-sensitive guanylyl cyclase inhibitor ODQ. In HUVECs, the phosphorylation of eNOS at Ser(1177) and increase in cytosolic NO level induced by nuciferine were mediated by extracellular Ca(2+) influx. Under endothelium-free conditions, nuciferine attenuated CaCl2-induced contraction in Ca(2+)-free depolarizing medium. In the absence of extracellular calcium, nuciferine relieved the vasoconstriction induced by phenylephrine and the addition of CaCl2. Nuciferine also suppressed Ca(2+) influx in Ca(2+)-free K(+)-containing solution in VSMCs.Conclusions and Implications: Nuciferine has a vasorelaxant effect via both endothelium-dependent and -independent mechanisms. These results suggest that nuciferine may have a therapeutic effect on vascular diseases associated with aberrant vasoconstriction. [ABSTRACT FROM AUTHOR]

Background and Purpose: Abnormal Ca(2+) metabolism has been involved in the pathogenesis of vascular dysfunction associated with oxidative stress. Here, we have investigated the actions of H2 O2 on store-operated Ca(2+) (SOC) entry in coronary arteries and assessed whether it is impaired in arteries from a rat model of metabolic syndrome.Experimental Approach: Simultaneous measurements of intracellular Ca(2+) concentration and contractile responses were made in coronary arteries from Wistar and obese Zucker rats, mounted in microvascular myographs, and the effects of H2 O2 were assessed.Key Results: H2 O2 raised intracellular Ca(2+) concentrations, accompanied by simultaneous vasoconstriction that was markedly reduced in a Ca(2+) -free medium. Upon Ca(2+) re-addition, a nifedipine-resistant sustained Ca(2+) entry, not coupled to contraction, was obtained in endothelium-denuded coronary arteries. The effect of H2 O2 on this voltage-independent Ca(2+) influx was concentration-dependent, and high micromolar H2 O2 concentrations were inhibitory and reduced SOC entry evoked by inhibition of the sarcoplasmic reticulum ATPase (SERCA). H2 O2 -induced increases in Fura signals were mimicked by Ba(2+) and reduced by heparin, Gd(3+) ions and by Pyr6, a selective inhibitor of the Orai1-mediated Ca(2+) entry,. In coronary arteries from obese Zucker rats, intracellular Ca(2+) mobilization and SOC entry activated by acute exposure to H2 O2 were augmented and associated with local oxidative stress.Conclusion and Implications: H2 O2 exerted dual concentration-dependent stimulatory/inhibitory effects on store-operated, IP3 receptor-mediated and Orai1-mediated Ca(2+) entry, not coupled to vasoconstriction in coronary vascular smooth muscle. SOC entry activated by H2 O2 was enhanced and associated with vascular oxidative stress in coronary arteries in metabolic syndrome. [ABSTRACT FROM AUTHOR]

Background and Purpose: Stimulation of the A1 adenosine receptor and angiotensin II receptor type-1 (AT1 receptor) causes vasoconstriction through activation of cytochrome P450 4A (CYP4A) and ERK1/2. Thus, we hypothesized that acute angiotensin II activation alters the vasomotor response induced by the non-selective adenosine receptor agonist, NECA, in mouse mesenteric arteries (MAs).Experimental Approach: We used a Danish Myo Technology wire myograph to measure muscle tension in isolated MAs from wild type (WT), A1 receptor and A2B receptor knockout (KO) mice. Western blots were performed to determine the expression of AT1 receptors and CYP4A.Key Results: Acute exposure (15 min) to angiotensin II attenuated the NECA-dependent vasodilatation and enhanced vasoconstriction. This vasoconstrictor effect of angiotensin II in NECA-treated MAs was abolished in A1 receptor KO mice and in WT mice treated with the A1 receptor antagonist DPCPX, CYP4A inhibitor HET0016 and ERK1/2 inhibitor PD98059. In MAs from A2B receptor KO mice, the vasoconstrictor effect of angiotensin II on the NECA-induced response was shown to be dependent on A1 receptors. Furthermore, in A2B receptor KO mice, the expression of AT1 receptors and CYP4A was increased and the angiotensin II-induced vasoconstriction enhanced. In addition, inhibition of KATP channels with glibenclamide significantly reduced NECA-induced vasodilatation in WT mice.Conclusions and Implications: Acute angiotensin II stimulation enhanced A1 receptor-dependent vasoconstriction and inhibited A2B receptor-dependent vasodilatation, leading to a net vasoconstriction and altered vasomotor response to NECA in MAs. This interaction may be important in the regulation of BP. [ABSTRACT FROM AUTHOR]

Background and Purpose In arterial smooth muscle cells (myocytes), intravascular pressure stimulates membrane depolarization and vasoconstriction (the myogenic response). Ion channels proposed to mediate pressure-induced depolarization include several transient receptor potential ( TRP) channels, including TRPM4, and transmembrane protein 16 A ( TMEM16 A), a Ca2+-activated Cl− channel (CaCC). 9-Phenanthrol, a putative selective TRPM4 channel inhibitor, abolishes myogenic tone in cerebral arteries, suggesting that either TRPM4 is essential for pressure-induced depolarization, upstream of activation of other ion channels or that 9-phenanthrol is non-selective. Here, we tested the hypothesis that 9-phenanthrol is also a TMEM16 A channel blocker, an ion channel for which few inhibitors have been identified. Experimental Approach Patch clamp electrophysiology was used to measure rat cerebral artery myocyte and human recombinant TMEM16 A ( rTMEM16 A) currents or currents generated by recombinant bestrophin-1, another Ca2+-activated Cl− channel, expressed in HEK293 cells. Key Results 9-Phenanthrol blocked myocyte TMEM16 A currents activated by either intracellular Ca2+ or Eact, a TMEM16 A channel activator. In contrast, 9-phenanthrol did not alter recombinant bestrophin-1 currents. 9-Phenanthrol reduced arterial myocyte TMEM16 A currents with an IC50 of ∼12 μM. Cell-attached patch recordings indicated that 9-phenanthrol reduced single rTMEM16 A channel open probability and mean open time, and increased mean closed time without affecting the amplitude. Conclusions and Implications These data identify 9-phenanthrol as a novel TMEM16 A channel blocker and provide an explanation for the previous observation that 9-phenanthrol abolishes myogenic tone when both TRPM4 and TMEM16 A channels contribute to this response. 9-Phenanthrol may be a promising candidate from which to develop TMEM16 A channel-specific inhibitors. [ABSTRACT FROM AUTHOR]

Background and Purpose Transient receptor potential vanilloid-4 ( TRPV4) is a calcium-permeant ion channel that is known to affect vascular function. The ability of TRPV4 to cause a vasoconstriction in blood vessels has not yet been mechanistically examined. Further in neuronal cells, TRPV4 signalling can be potentiated by GPCR activation. Thus, we studied the mechanisms underlying the vascular contractile action of TRPV4 and the GPCR-mediated potentiation of such vasoconstriction, both of which are as yet unappreciated aspects of TRPV4 function. Experimental Approach The mechanisms of TRPV4-dependent regulation of vascular tone in isolated mouse aortae were studied using wire myography. TRPV4-dependent calcium signalling and prostanoid production was studied in cultured human umbilical vein endothelial cells (HUVECs). Key Results In addition to the well-documented vasorelaxation response triggered by TRPV4 activation, we report here a TRPV4-triggered vasoconstriction in the mouse aorta that involves a COX-generated Tx receptor ( TP) agonist that acts in a MAPK and Src kinase signalling dependent manner. This constriction is potentiated by activation of the GPCRs for angiotensin ( AT1 receptors) or proteinases ( PAR1 and PAR2) via transactivation of the EGF receptor and a process involving PKC. TRPV4-dependent vascular contraction can be blocked by COX inhibitors or with TP antagonists. Further, TRPV4 activation in HUVECs stimulated Tx release as detected by an elisa. Conclusion and Implications We conclude that the GPCR potentiation of TRPV4 action and TRPV4-dependent Tx receptor activation are important regulators of vascular function and could be therapeutically targeted in vascular diseases. [ABSTRACT FROM AUTHOR]

Background and Purpose We assessed whether endothelin-1 ( ET-1) inhibits NO and contributes to endothelial dysfunction in penile arteries in a model of insulin resistance-associated erectile dysfunction ( ED). Experimental Approach Vascular function was assessed in penile arteries, from obese ( OZR) and lean ( LZR) Zucker rats, mounted in microvascular myographs. Changes in basal and stimulated levels of superoxide ( O2−) were detected by lucigenin-enhanced chemiluminescence and ET receptor expression was determined by immunohistochemistry. Key Results ET-1 stimulated acute O2− production that was blunted by tempol and the NADPH oxidase inhibitor, apocynin, but markedly enhanced in obese animals. ET-1 inhibited the vasorelaxant effects of ACh and of the NO donor S- nitroso- N-acetyl- DL-penicillamine in arteries from both LZR and OZR. Selective ETA ( BQ123) or ETB receptor ( BQ788) antagonists reduced both basal and ET-1-stimulated superoxide generation and reversed ET-1-induced inhibition of NO-mediated relaxations in OZR, while only BQ-123 antagonized ET-1 actions in LZR. ET-1-induced vasoconstriction was markedly enhanced by NO synthase blockade and reduced by endothelium removal and apocynin. In endothelium-denuded penile arteries, apocynin blunted augmented ET-1-induced contractions in OZR. Both ETA and ETB receptors were expressed in smooth muscle and the endothelial layer and up-regulated in arteries from OZR. Conclusions and Implications ET-1 stimulates ETA-mediated NADPH oxidase-dependent ROS generation, which inhibits endothelial NO bioavailability and contributes to ET-1-induced contraction in healthy penile arteries. Enhanced vascular expression of ETB receptors contributes to augmented ROS production, endothelial dysfunction and increased vasoconstriction in erectile tissue from insulin-resistant obese rats. Hence, antagonism of ETB receptors might improve the ED associated with insulin-resistant states. [ABSTRACT FROM AUTHOR]

Background and Purpose The α1-adrenoceptor family plays a critical role in regulating ocular perfusion by mediating responses to catecholamines. The purpose of the present study was to determine the contribution of individual α1-adrenoceptor subtypes to adrenergic vasoconstriction of retinal arterioles using gene-targeted mice deficient in one of the three adrenoceptor subtypes (α1 A- AR−/−, α1 B- AR−/− and α1 D- AR−/− respectively). Experimental Approach Using real-time PCR, m RNA expression for individual α1-adrenoceptor subtypes was determined in murine retinal arterioles. To assess the functional relevance of the three α1-adrenoceptor subtypes for mediating vascular responses, retinal vascular preparations from wild-type mice and mice deficient in individual α1-adrenoceptor subtypes were studied in vitro using video microscopy. Key Results Retinal arterioles expressed m RNA for all three α1-adrenoceptor subtypes. In functional studies, arterioles from wild-type mice with intact endothelium responded only negligibly to the α1-adrenoceptor agonist phenylephrine. In endothelium-damaged arterioles from wild-type mice, phenylephrine evoked concentration-dependent constriction that was attenuated by the α1-adrenoceptor blocker prazosin. Strikingly, phenylephrine only minimally constricted endothelium-damaged retinal arterioles from α1 B- AR−/− mice, whereas arterioles from α1 A- AR−/− and α1 D- AR−/− mice constricted similarly to arterioles from wild-type mice. Constriction to U46619 was similar in endothelium-damaged retinal arterioles from all four mouse genotypes. Conclusions and Implications The present study is the first to demonstrate that α1-adrenoceptor-mediated vasoconstriction in murine retinal arterioles is buffered by the endothelium. When the endothelium is damaged, a vasoconstricting role of the α1 B-adrenoceptor subtype is unveiled. Hence, the α1 B-adrenoceptor may represent a target to selectively modulate retinal blood flow in ocular diseases associated with endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Background and Purpose Pulmonary arteries ( PAs) are innervated, but little is known about the role of neuronal axis in pulmonary hypertension ( PH). Here, we have examined the role of the neuropeptide Y ( NPY) and its Y1 receptor in PH pathogenesis. Experimental Approach NPY was localized by immunofluorescence. Expression of NPY and Y1 receptor were determined by quantitative PCR. Cellular response to NPY stimulation was assessed by Western blotting, thymidine incorporation and calcium imaging. Wire myography and isolated perfused mouse lung were applied to study pulmonary vasoactive effects of NPY. Selective receptor antagonists were used to assess the contribution of receptor subtypes in mediating NPY effects. Key Results Samples from PH patients showed increased NPYergic innervation within the PA wall and higher Y1 receptor expression, compared with donors. However, NPY levels were unchanged in both PA and serum. In the chronic hypoxic mouse model, Y1 receptor were up-regulated, while expression of both NPY and Y1 receptor was increased in the lungs of monocrotaline and SU5416-hypoxia rats. On a functional level, NPY acutely increased intracellular calcium levels and enhanced vasoconstriction of lung vessels preconstricted with adrenaline. Furthermore, NPY stimulated proliferation of human pulmonary arterial smooth muscle cells and activated p38 and PKD pathways. Correspondingly, higher phosphorylation of PKD was observed in remodelled vessels from PH patients. The selective Y1 receptor antagonist, BIBO 3304, concentration-dependently inhibited vasoconstrictive and proliferative effects of NPY. Conclusions and Implications NPY and Y1 receptor are possible mediators of both vasoconstriction and pulmonary vascular remodelling in PH. [ABSTRACT FROM AUTHOR]

Background and Purpose Because angiotensin- II-mediated porcine coronary artery ( PCA) vasoconstriction is blocked by protein tyrosine kinase ( PYK) inhibitors, we hypothesized that proteinase-activated receptors ( PARs), known to regulate vascular tension, like angiotensin- II, would also cause PCA contractions via PYK-dependent signalling pathways. Experimental Approach Contractions of intact and endothelium-free isolated PCA rings, stimulated by PAR1/ PAR2-activating peptides, angiotensin- II, PGF2α, EGF, PDGF and KCl, were monitored with/without multiple signalling pathway inhibitors, including AG-tyrphostins AG18 (non-specific PYKs), AG1478 ( EGF-receptor kinase), AG1296 ( PDGF receptor kinase), PP1 ( Src kinase), U0126 and PD98059 ( MEK/ MAPKinase kinase), indomethacin/ SC-560/ NS-398 ( COX-1/2) and L- NAME ( NOS). Key Results AG18 inhibited the contractions induced by all the agonists except KCl, whereas U0126 attenuated contractions induced by PAR1/ PAR2 agonists, EGF and angiotensin- II, but not by PGF2α, the COX-produced metabolites of arachidonate and KCl. PP1 only affected the responses to PAR1/ PAR2-activating peptides and angiotensin- II. The EGF-kinase inhibitor, AG1478, attenuated contractions initiated by the PARs ( PAR2 >> PAR1) and EGF itself, but not by angiotensin- II, PGF2α or KCl. COX-1/2 inhibitors blocked the contractions induced by all the agonists, except KCl and PGF2α. Conclusion and Implications PAR1/2-mediated contractions of the PCA are dependent on Src and MAPKinase and, in part, involve EGF-receptor-kinase transactivation and the generation of a COX-derived contractile agonist. However, the PYK signalling pathways used by PARs are distinct from each other and from those triggered by angiotensin- II and EGF. These signalling pathways may be therapeutic targets for managing coagulation-proteinase-induced coronary vasospasm. [ABSTRACT FROM AUTHOR]

Application of cannabinoids and endocannabinoids to perfused vascular beds or individual isolated arteries results in changes in vascular resistance. In most cases, the result is vasorelaxation, although vasoconstrictor responses are also observed. Cannabinoids also modulate the actions of vasoactive compounds including acetylcholine, methoxamine, angiotensin II and U46619 (thromboxane mimetic). Numerous mechanisms of action have been proposed including receptor activation, potassium channel activation, calcium channel inhibition and the production of vasoactive mediators such as calcitonin gene-related peptide, prostanoids, NO, endothelial-derived hyperpolarizing factor and hydrogen peroxide. The purpose of this review is to examine the evidence for the range of receptors now known to be activated by cannabinoids. Direct activation by cannabinoids of CB1, CBe, TRPV1 (and potentially other TRP channels) and PPARs in the vasculature has been observed. A potential role for CB2, GPR55 and 5- HT1 A has also been identified in some studies. Indirectly, activation of prostanoid receptors ( TP, IP, EP1 and EP4) and the CGRP receptor is involved in the vascular responses to cannabinoids. The majority of this evidence has been obtained through animal research, but recent work has confirmed some of these targets in human arteries. Vascular responses to cannabinoids are enhanced in hypertension and cirrhosis, but are reduced in obesity and diabetes, both due to changes in the target sites of action. Much further work is required to establish the extent of vascular actions of cannabinoids and the application of this research in physiological and pathophysiological situations. Linked Articles This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit [ABSTRACT FROM AUTHOR]

Background and Purpose: The P2Y₁₄ receptor is the newest member of the P2Y receptor family; it is G(i/o) protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5'-diphosphoglucose) (7-10-fold more potent than UDP-glucose). This study investigated whether P2Y₁₄ receptors were functionally expressed in porcine isolated pancreatic arteries.Experimental Approach: Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA₂ mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit.Key Results: Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y₁₄ receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y₆ receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA₂, PGF(2α) and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN.Conclusions and Implications: P2Y₁₄ receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca²⁺ levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA₂, PGF(2α) and endothelin-1. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE The P2Y14 receptor is the newest member of the P2Y receptor family; it is Gi/o protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5'-diphosphoglucose) (7-10-fold more potent than UDP-glucose). This study investigated whether P2Y14 receptors were functionally expressed in porcine isolated pancreatic arteries. EXPERIMENTAL APPROACH Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA2 mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit. KEY RESULTS Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) ≥ UDP-glucose = UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y14 receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y6 receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA2, PGF2α and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN. CONCLUSIONS AND IMPLICATIONS P2Y14 receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca2+ levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA2, PGF2α and endothelin-1. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. EXPERIMENTAL APPROACH Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPa and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. KEY RESULTS The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. CONCLUSIONS AND IMPLICATIONS In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity. [ABSTRACT FROM AUTHOR]

Background and Purpose Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 ( ET-1) causes vasoconstriction via endothelin receptor type A ( ETAR), but could activate ETBR in the endothelium and release vasodilator substances. However, the roles of ETBR in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear. Experimental Approach Pressurized mesenteric microvessels from pregnant and virgin Sprague- Dawley rats were loaded with fura-2/ AM for simultaneous measurement of diameter and [ Ca2+]i. Key Results High KCl (51 mM) and phenylephrine (PHE) caused increases in vasoconstriction and [ Ca2+]i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [ Ca2+]i. Pretreatment with the ETBR antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. ACh caused endothelium-dependent relaxation and decreased [ Ca2+]i, and was more potent in pregnant than in virgin rats. ET-1 + ETAR antagonist BQ-123, and the ETBR agonists sarafotoxin 6c ( S6c) and IRL-1620 caused greater vasodilation in pregnant than in virgin rats with no changes in [ Ca2+]i, suggesting up-regulated ETBR-mediated relaxation pathways. ACh-, S6c- and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor Nω-nitro- l-arginine methyl ester, and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ETBR in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ETBR. Conclusions and Implications The enhanced ETBR-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy. [ABSTRACT FROM AUTHOR]

Background and Purpose 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. Experimental Approach 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. Key Results 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. Conclusions and Implications Arginase inhibition alleviates hypertension in metabolic syndrome directly through endothelial-dependent relaxation/ NO signalling protection and indirectly through inhibition of insulin resistance and hypertriglyceridaemia. [ABSTRACT FROM AUTHOR]

Recent advances have improved our understanding of the renin-angiotensin system ( RAS). These have included the recognition that angiotensin ( Ang)-(1-7) is a biologically active product of the RAS cascade. The identification of the ACE homologue ACE2, which forms Ang-(1-7) from Ang II, and the GPCR Mas as an Ang-(1-7) receptor have provided the necessary biochemical and molecular background and tools to study the biological significance of Ang-(1-7). Most available evidence supports a counter-regulatory role for Ang-(1-7) by opposing many actions of Ang II on AT1 receptors, especially vasoconstriction and proliferation. Many studies have now shown that Ang-(1-7) by acting via Mas receptor exerts inhibitory effects on inflammation and on vascular and cellular growth mechanisms. Ang-(1-7) has also been shown to reduce key signalling pathways and molecules thought to be relevant for fibrogenesis. Here, we review recent findings related to the function of the ACE2/ Ang-(1-7)/ Mas axis and focus on the role of this axis in modifying processes associated with acute and chronic inflammation, including leukocyte influx, fibrogenesis and proliferation of certain cell types. More attention will be given to the involvement of the ACE2/ Ang-(1-7)/ Mas axis in the context of renal disease because of the known relevance of the RAS for the function of this organ and for the regulation of kidney inflammation and fibrosis. Taken together, this knowledge may help in paving the way for the development of novel treatments for chronic inflammatory and renal diseases. [ABSTRACT FROM AUTHOR]

Background and Purpose Recent evidence suggested that urotensin II ( UII) and its paralog peptide UII-related peptide ( URP) might exert common but also divergent physiological actions. Unfortunately, none of the existing antagonists were designed to discriminate specific UII- or URP-associated actions, and our understanding, on how these two endogenous peptides can trigger different, but also common responses, is limited. Experimental Approach Ex vivo rat and monkey aortic ring contraction as well as dissociation kinetics studies using transfected CHO cells expressing the human urotensin ( UT) receptors were used in this study. Key Results Ex vivo rat and monkey aortic ring contraction studies revealed the propensity of [ Pep4] URP to decrease the maximal response of human UII ( hUII) without any significant change in potency, whereas no effect was noticeable on the URP-induced vasoconstriction. Dissociation experiments demonstrated the ability of [ Pep4] URP to increase the dissociation rate of hUII, but not URP. Surprisingly, URP, an equipotent UII paralog, was also able to accelerate the dissociation rate of membrane-bound 125I-hUII, whereas hUII had no noticeable effect on URP dissociation kinetics. Further experiments suggested that an interaction between the glutamic residue at position 1 of hUII and the UT receptor seems to be critical to induce conformational changes associated with agonistic activation. Finally, we demonstrated that the N-terminal domain of the rat UII isoform was able to act as a specific antagonist of the URP-associated actions. Conclusion Such compounds, that is [ Pep4] URP and rUII(1-7), should prove to be useful as new pharmacological tools to decipher the specific role of UII and URP in vitro but also in vivo. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE 5-HT is taken up by and stored in adrenergic nerves and periarterial nerve stimulation (PNS) releases 5-HT to cause vasoconstriction in rat mesenteric arteries. The present study investigated whether PNS-released 5-HT stored in adrenergic nerves affects the function of perivascular calcitonin gene-related peptide-containing (CGRPergic) nerves. EXPERIMENTAL APPROACH Rat mesenteric vascular beds without endothelium and with active tone were perfused with Krebs solution. Changes in perfusion pressure in response to PNS and CGRP injection were measured before (control) and after perfusion of Krebs solution containing 5-HT (10 µM) for 20 min. Distributions of 5-HT- and TH-immunopositive fibres in mesenteric arteries were studied using immunohistochemical methods. KEY RESULTS PNS (1-4 Hz) frequency dependently caused adrenergic nerve-mediated vasoconstriction followed by CGRPergic nerve-mediated vasodilatation. 5-HT treatment inhibited PNS-induced vasodilatation without affecting exogenous CGRP-induced vasodilatation, while it augmented PNS-induced vasoconstriction. Guanethidine (adrenergic neuron blocker), methysergide (non-selective 5-HT receptor antagonist) and BRL15572 (selective 5-HT1D receptor antagonist) abolished inhibition of PNS-induced vasodilatation in 5-HT-treated preparations. Combined treatment with 5-HT and desipramine (catecholamine transporter inhibitor), but not fluoxetine (selective 5-HT reuptake inhibitor), did not inhibit PNS-induced vasodilatation. Exogenous 5-HT inhibited PNS-induced vasodilatation, which was antagonized by methysergide. In immunohistochemical experiments, 5-HT-immunopositive nerves, colocalized with adrenergic TH-immunopositive nerves, were observed only in 5-HT-treated mesenteric arteries, but not in control preparations or arteries co-treated with desipramine. CONCLUSIONS AND IMPLICATIONS These results suggest that 5-HT can be taken up by and released from adrenergic nerves in vitro by PNS to inhibit CGRPergic nerve transmission in rat mesenteric arteries. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) causes long-lasting vasoconstrictions. These can be prevented by ETA receptor antagonists but are only poorly reversed by these drugs. We tested the hypothesis that endothelin ETA receptors are susceptible to allosteric modulation by endogenous agonists and exogenous ligands. EXPERIMENTAL APPROACH Rat isolated mesenteric resistance arteries were pretreated with capsaicin and studied in wire myographs, in the presence of L-NAME and indomethacin to concentrate on arterial smooth muscle responses. KEY RESULTS Endothelins caused contractions with equal maximum but differing potency (ET-1 = ET-2 > ET-3). ET-11-15 neither mimicked nor antagonized these effects in the absence and presence of ET16-21. 4AlaET-1 (ETB agonist) and BQ788 (ETB antagonist) were without effects. BQ123 (peptide ETA antagonist) reduced the sensitivity and relaxed the contractile responses to endothelins. Both effects depended on the agonist (pKB: ET-3 = ET-1 > ET-2; % relaxation: ET-3 = ET-2 > ET-1). Also, with PD156707 (non-peptide ETA antagonist) agonist-dependence and a discrepancy between preventive and inhibitory effects were observed. The latter was even more marked with bulky analogues of BQ123 and PD156707. CONCLUSIONS AND IMPLICATIONS These findings indicate allosteric modulation of arterial smooth muscle ETA receptor function by endogenous agonists and by exogenous endothelin receptor antagonists. This may have consequences for the diagnosis and pharmacotherapy of diseases involving endothelins. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE FTY720 (Fingolimod) is a recently approved orally administered drug for the treatment of multiple sclerosis. Phase II and III clinical trials have demonstrated that this drug modestly increases BP. We previously showed that inhibition of sphingosine kinase increases vascular tone and BP in hypertensive, but not normotensive rats. Since FTY720 is reported to have inhibitory effects on sphingosine kinase, we investigated whether FTY720 increases vascular tone and BP only in hypertensive rats via this mechanism. EXPERIMENTAL APPROACH The contractile and BP modulating effects of FTY720 were studied in vivo and ex vivo (wire myography) in age-matched normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). KEY RESULTS Oral administration of FTY720 induced an increase in mean arterial pressure in SHR, whereas a decrease in BP was observed in WKY rats, as measured 24 h after administration. Similar to the sphingosine kinase inhibitor dimethylsphingosine (DMS), FTY720 induced large contractions in isolated carotid arteries from SHR, but not in those from WKY. In contrast, the phosphorylated form of FTY720 did not induce contractions in isolated carotid arteries from SHR. FTY720-induced contractions were inhibited by endothelium denudation, COX and thromboxane synthase inhibitors, and by thromboxane receptor antagonism, indicating that (like DMS-induced contractions) they were endothelium-dependent and mediated by thromboxane A2. CONCLUSIONS AND IMPLICATIONS These data demonstrate that FTY720 increases vascular tone and BP only in hypertensive rats, most likely as a result of its inhibitory effect on sphingosine kinase. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE TASK1 (K2P3.1) two-pore-domain K+ channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1. EXPERIMENTAL APPROACH Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes. KEY RESULTS ET-1 inhibited TASK1-mediated IKN currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ETA (IC50= 0.08 nM) and ETB (IC50= 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser336 and Ser393. Mutation of Ser393 rendered TASK1 channels insensitive to ETA- or ETB-mediated current inhibition. In contrast, removal of Ser336 selectively attenuated ETA-dependent TASK1 regulation without affecting the ETB pathway. CONCLUSIONS AND IMPLICATIONS ET-1 regulated vascular TASK1 currents through ETA and ETB receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1. EXPERIMENTAL APPROACH SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24 h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O2•−) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively. KEY RESULTS I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ETB mRNA expression, and down-regulated ETA mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ETB receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ETB receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ETB receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, Nω-nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O2•− production by increasing NADPH oxidase 1 (NOX-1) and p47phox mRNA. CONCLUSIONS AND IMPLICATIONS Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ETB receptors and decrease in NO bioavailability. [ABSTRACT FROM AUTHOR]

Obesity has become a serious global health issue affecting both adults and children. Recent devolopments in world demographics and declining health status of the world's population indicate that the prevalence of obesity will continue to increase in the next decades. As a disease, obesity has deleterious effects on metabolic homeostasis, and affects numerous organ systems including heart, kidney and the vascular system. Thus, obesity is now regarded as an independent risk factor for atherosclerosis-related diseases such as coronary artery disease, myocardial infarction and stroke. In the arterial system, endothelial cells are both the source and target of factors contributing to atherosclerosis. Endothelial vasoactive factors regulate vascular homeostasis under physiological conditions and maintain basal vascular tone. Obesity results in an imbalance between endothelium-derived vasoactive factors favouring vasoconstriction, cell growth and inflammatory activation. Abnormal regulation of these factors due to endothelial cell dysfunction is both a consequence and a cause of vascular disease processes. Finally, because of the similarities of the vascular pathomechanisms activated, obesity can be considered to cause accelerated, 'premature' vascular aging. Here, we will review some of the pathomechanisms involved in obesity-related activation of endothelium-dependent vasoconstriction, the clinical relevance of obesity-associated vascular risk, and therapeutic interventions using 'endothelial therapy' aiming at maintaining or restoring vascular endothelial health. LINKED ARTICLES This article is part of a themed section on Fat and Vascular Responsiveness. To view the other articles in this section visit [ABSTRACT FROM AUTHOR]

Background and Purpose: Antenatal nicotine exposure causes aberrant vascular reactivity and increased blood pressure in adult male rat offspring in a sex-dependent manner. The present study tested the hypothesis that maternal nicotine administration increases the production of reactive oxygen species resulting in the vascular hypertensive reactivity in male offspring.Experimental Approach: Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps throughout the gestation. The vascular oxidative damage and dysfunction were determined in 5-month-old male offspring. Contraction studies were performed on isolated aortas and their expression of NADPH oxidase (Nox2)/gp91 and nox4 determined by Western blot analysis. In addition, oxidative damage in the vessel wall was determined by measuring malondialdehyde concentrations, vascular superoxide production and SOD activity.Key Results: Antenatal nicotine significantly increased angiotensin II-induced arterial contractions in the offspring. The exaggerated vascular contractions were inhibited by both apocynin (a Nox inhibitor) and tempol (a SOD mimetic) in a concentration-dependent manner. In addition, ACh-induced relaxations were impaired in aortas isolated from the nicotine-treated offspring, which were restored by both apocynin and tempol in a concentration-dependent manner. The nicotine treatment significantly decreased the superoxide dismutase activity and increased malondialdehyde, superoxide and nitrotyrosine protein levels in the vascular wall. Consistently, antenatal nicotine exposure significantly enhanced the protein expression of NADPH oxidase Nox2/gp91, but not Nox4 in the aorta.Conclusions and Implications: The present findings suggest that antenatal nicotine exposure results in the programming of heightened oxidative stress and vascular hypertensive reactivity via a Nox2-dependent mechanism, leading to an increased risk of hypertension in adult offspring. [ABSTRACT FROM AUTHOR]

Endothelium-dependent contractions contribute to endothelial dysfunction in various animal models of aging, diabetes and cardiovascular diseases. In the spontaneously hypertensive rat, the archetypal model for endothelium-dependent contractions, the production of the endothelium-derived contractile factors (EDCF) involves an increase in endothelial intracellular calcium concentration, the production of reactive oxygen species, the predominant activation of cyclooxygenase-1 (COX-1) and to a lesser extent that of COX-2, the diffusion of EDCF towards the smooth muscle cells and the subsequent stimulation of their thromboxane A2-endoperoxide TP receptors. Endothelium-dependent contractions are also observed in various models of hypertension, aging and diabetes. They generally also involve the generation of COX-1- and/or COX-2-derived products and the activation of smooth muscle TP receptors. Depending on the model, thromboxane A(2), PGH(2), PGF(2α), PGE(2) and paradoxically PGI(2) can all act as EDCFs. In human, the production of COX-derived EDCF is a characteristic of the aging and diseased blood vessels, with essential hypertension causing an earlier onset and an acceleration of this endothelial dysfunction. As it has been observed in animal models, COX-1, COX-2 or both isoforms can contribute to these endothelial dysfunctions. Since in most cases, the activation of TP receptors is the common downstream effector, selective antagonists of this receptor should curtail endothelial dysfunction and be of therapeutic interest in the treatment of cardiovascular disorders. [ABSTRACT FROM AUTHOR]

Background and Purpose: Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone.Experimental Approach: We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP.Key Results: Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation.Conclusions and Implications: TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. [ABSTRACT FROM AUTHOR]