Structural identification of vasodilator binding sites on the SUR2 subunit.

ATP-sensitive potassium channels (KATP), composed of Kir6 and SUR subunits, convert the metabolic status of the cell into electrical signals. Pharmacological activation of SUR2- containing KATP channels by class of small molecule drugs known as KATP openers leads to hyperpolarization of excitable cells and to vasodilation. Thus, KATP openers could be used to treat cardiovascular diseases. However, where these vasodilators bind to KATP and how they activate the channel remains elusive. Here, we present cryo-EM structures of SUR2A and SUR2B subunits in complex with Mg-nucleotides and P1075 or levcromakalim, two chemically distinct KATP openers that are specific to SUR2. Both P1075 and levcromakalim bind to a common site in the transmembrane domain (TMD) of the SUR2 subunit, which is between TMD1 and TMD2 and is embraced by TM10, TM11, TM12, TM14, and TM17. These KATP openers synergize with Mg-nucleotides to stabilize SUR2 in the NBD-dimerized occluded state to activate the channel.

Target-responsive vasoactive probes for ultrasensitive molecular imaging.

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.

Aberrant expression of calcitonin gene-related peptide and its correlation with prognosis in severe childhood pneumonia

OBJECTIVES: The purpose of this study was to evaluate the relationship between the serum levels of calcitonin gene-related peptide (CGRP) and the prognosis of pediatric patients with severe pneumonia. METHODS: Children diagnosed with severe pneumonia (n=76) were stratified into the survival (n=58) and non-survival groups (n=18) according to their 28-day survival status and into the non-risk (n=51), risk (n=17) and high-risk (n=8) categories based on the pediatric critical illness score (PCIS). Demographic data and laboratory results were collected. Serum CGRP levels were determined by enzyme-linked immunosorbent assay (ELISA). A receiver operating characteristic (ROC) curve was generated to determine the cutoff score for high CGRP levels. RESULTS: Serum CGRP levels were significantly higher in the survival group than in the non-survival group and were significantly higher in the non-risk group than in the risk and high-risk groups. The ROC curve for the prognostic potential of CGRP yielded a significant area under the curve (AUC) value with considerable sensitivity and specificity. CONCLUSION: Our findings show that CGRP downregulation might be a diagnostic marker that predicts the prognosis and survival of children with severe pneumonia.

The Inhibition of CD39 and CD73 Cell Surface Ectonucleotidases by Small Molecular Inhibitors Enhances the Mobilization of Bone Marrow Residing Stem Cells by Decreasing the Extracellular Level of Adenosine.

We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.

Novel Anti-inflammatory and Vasodilatory ω-3 Endocannabinoid Epoxide Regioisomers.

Accumulating evidence suggests that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) offer protection against vascular inflammation, neuroinflammation, hypertension, and thrombosis. Recently, biochemical studies have demonstrated that these benefits are partially mediated by their conversion to ω-3 endocannabinoid epoxide metabolites. These lipid metabolites originate from the epoxidation of ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by cytochrome P450 (CYP) epoxygenases to form epoxydocosapentaenoic acid-ethanolamides (EDP-EAs) and epoxyeicosatetraenoic acid-ethanolamides (EEQ-EAs), respectively. The EDP-EAs and EEQ-EAs are endogenously produced in rat brain and peripheral organs. Additionally, EDP-EAs and EEQ-EAs dose-dependently decrease pro-inflammatory IL-6 cytokine and increased anti-inflammatory IL-10 cytokine. Furthermore, the EEQ-EAs and EDP-EAs attenuate angiogenesis and cell migration in cancer cells, induce vasodilation in bovine coronary arteries, and reciprocally regulate platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides represent a new class of dual acting molecules that display unique pharmacological properties.

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.

The Effect of Dexmedetomidine on Oral Mucosal Blood Flow and the Absorption of Lidocaine.

Dexmedetomidine (DEX) is a sedative and analgesic agent that acts via the alpha-2 adrenoreceptor and is associated with reduced anesthetic requirements, as well as attenuated blood pressure and heart rate in response to stressful events. A previous study reported that cat gingival blood flow was controlled via sympathetic alpha-adrenergic fibers involved in vasoconstriction. In the present study, experiment 1 focused on the relationship between the effects of DEX on alpha adrenoreceptors and vasoconstriction in the tissues of the oral cavity and compared the palatal mucosal blood flow (PMBF) in rabbits between general anesthesia with sevoflurane and sedation with DEX. We found that the PMBF was decreased by DEX presumably because of the vasoconstriction of oral mucosal vessels following alpha-2 adrenoreceptor stimulation by DEX. To assess if this vasoconstriction would allow decreased use of locally administered epinephrine during DEX infusion, experiment 2 in the present study monitored the serum lidocaine concentration in rabbits to compare the absorption of lidocaine without epinephrine during general anesthesia with sevoflurane and sedation with DEX. The depression of PMBF by DEX did not affect the absorption of lidocaine. We hypothesize that this is because lidocaine dilates the blood vessels, counteracting the effects of DEX. In conclusion, despite decreased palatal blood flow with DEX infusion, local anesthetics with vasoconstrictors should be used in implant and oral surgery even with administered DEX.

Mercaptopyruvate acts as endogenous vasodilator independently of 3-mercaptopyruvate sulfurtransferase activity.

Hydrogen sulfide (H2S) is produced by the action of cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H2S and pyruvate. H2S is recognized as an endogenous gaseous mediator with multiple regulatory roles in mammalian cells and organisms. In the present study we demonstrate that MPT, the endogenous substrate of 3-MST, acts also as endogenous H2S donor. Colorimetric, amperometric and fluorescence based assays demonstrated that MPT releases H2S in vitro in an enzyme-independent manner. A functional study was performed on aortic rings harvested from C57BL/6 (WT) or 3-MST-knockout (3-MST-/-) mice with and without endothelium. MPT relaxed mouse aortic rings in endothelium-independent manner and at the same extent in both WT and 3-MST-/- mice. N5-(1-Iminoethyl)-l-ornithine dihydrochloride (L-NIO, an inhibitor of endothelial nitric oxide synthase) as well as 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor) did not affect MPT relaxant action. Conversely, hemoglobin (as H2S scavenger), as well as glybenclamide (an ATP-dependent potassium channel blocker) markedly reduced MPT-induced relaxation. The functional data clearly confirmed a non enzymatic vascular effect of MPT. In conclusion, MPT acts also as an endogenous H2S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to increase H2S in conditions where H2S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease.

Induction of microRNA-199 by Nitric Oxide in Endothelial Cells Is Required for Nitrovasodilator Resistance via Targeting of Prostaglandin I2 Synthase.

BACKGROUND: Nitrates are widely used to treat coronary artery disease, but their therapeutic value is compromised by nitrate tolerance, because of the dysfunction of prostaglandin I2 synthase (PTGIS). MicroRNAs repress target gene expression and are recognized as important epigenetic regulators of endothelial function. The aim of this study was to determine whether nitrates induce nitrovasodilator resistance via microRNA-dependent repression of PTGIS gene expression. METHODS: Nitrovasodilator resistance was induced by nitroglycerin (100 mg·kg-1·d-1, 3 days) infusion in Apoe-/- mice. The responses of aortic arteries to nitric oxide donors were assessed in an organ chamber. The expression levels of microRNA-199 (miR-199)a/b were assayed by quantitative reverse transcription polymerase chain reaction or fluorescent in situ hybridization. RESULTS: In cultured human umbilical vein endothelial cells, nitric oxide donors induced miR-199a/b endogenous expression and downregulated PTGIS gene expression, both of which were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt or silence of serum response factor. Evidence from computational and luciferase reporter gene analyses indicates that the seed sequence of 976 to 982 in the 3'-untranslated region of PTGIS mRNA is a target of miR-199a/b. Gain functions of miR-199a/b resulting from chemical mimics or adenovirus-mediated overexpression increased PTGIS mRNA degradation in HEK293 cells and human umbilical vein endothelial cells. Furthermore, nitroglycerin-decreased PTGIS gene expression was prevented by miR-199a/b antagomirs or was mirrored by the enforced expression of miR-199a/b in human umbilical vein endothelial cells. In Apoe-/- mice, nitroglycerin induced the ectopic expression of miR-199a/b in the carotid arterial endothelium, decreased PTGIS gene expression, and instigated nitrovasodilator resistance, all of which were abrogated by miR-199a/b antagomirs or LNA-anti-miR-199. It is important that the effects of miR-199a/b inhibitions were abolished by adenovirus-mediated PTGIS deficiency. Moreover, the enforced expression of miR-199a/b in vivo repressed PTGIS gene expression and impaired the responses of aortic arteries to nitroglycerin/sodium nitroprusside/acetylcholine/cinaciguat/riociguat, whereas the exogenous expression of the PTGIS gene prevented nitrovasodilator resistance in Apoe-/- mice subjected to nitroglycerin infusion or miR-199a/b overexpression. Finally, indomethacin, iloprost, and SQ29548 improved vasorelaxation in nitroglycerin-infused Apoe-/- mice, whereas U51605 induced nitrovasodilator resistance. In humans, the increased expressions of miR-199a/b were closely associated with nitrate tolerance. CONCLUSIONS: Nitric oxide-induced ectopic expression of miR-199a/b in endothelial cells is required for nitrovasodilator resistance via the repression of PTGIS gene expression. Clinically, miR-199a/b is a novel target for the treatment of nitrate tolerance.

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols.

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 µM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC.

ANRIL regulates production of extracellular matrix proteins and vasoactive factors in diabetic complications.

noncoding RNAs (lncRNAs) have gained widespread interest due to their prevailing presence in various diseases. lncRNA ANRIL (a. k. a. CDKN2B-AS1) is located on human chromosome 9 (p21.3) and transcribed in opposite direction to the INK4b-ARF-INK4a gene cluster. It has been identified as a highly susceptible region for diseases such as coronary artery diseases and type 2 diabetes. Here, we explored its regulatory role in diabetic nephropathy (DN) and diabetic cardiomyopathy (DCM) in association with epigenetic modifiers p300 and polycomb repressive complex 2 (PRC2) complex. We used an ANRIL-knockout (ANRILKO) mouse model for this study. The wild-type and ANRILKO animals with or without streptozotocin-induced diabetes were monitored for 2 min. At the end of the time point, urine and tissues were collected. The tissues were measured for fibronectin (FN), type IV collagen (Col1α4), and VEGF mRNA and protein expressions. Renal function was determined by the measurement of 24-h urine volume and albumin/creatinine ratio at euthanasia. Renal and cardiac structures were investigated using periodic acid-Schiff stain and/or immunohistochemical analysis. Elevated expressions of extracellular matrix (ECM) proteins were prevented in ANRILKO diabetic animals. Furthermore, ANRILKO had a protective effect on diabetic mouse kidneys, as evidenced by lowering of urine volume and urine albumin levels in comparison with the wild-type diabetic animals. These alterations regulated by ANRIL may be mediated by p300 and enhancer of zeste 2 (EZH2) of the PRC2 complex. Our study concludes that ANRIL regulates functional and structural alterations in the kidneys and hearts in diabetes through controlling the expressions of ECM proteins and VEGF.

Angiotensin II-derived constrained peptides with antiplasmodial activity and suppressed vasoconstriction.

Angiotensin II (Ang II) is a natural mammalian hormone that has been described to exhibit antiplasmodial activity therefore constituting a promising alternative for the treatment of malaria. Despite its promise, the development of Ang II as an antimalarial is limited by its potent induction of vasoconstriction and its rapid degradation within minutes. Here, we used peptide design to perform targeted chemical modifications to Ang II to generate conformationally restricted (disulfide-crosslinked) peptide derivatives with suppressed vasoconstrictor activity and increased stability. Designed constrained peptides were synthesized chemically and then tested for antiplasmodial activity. Two lead constrained peptides were identified (i.e., peptides 1 and 2), each composed of 10 amino acid residues. These peptides exhibited very promising activity in both our Plasmodium gallinaceum (>80%) and Plasmodium falciparum (>40%) models, an activity that was equivalent to that of Ang II, and led to complete suppression of vasoconstriction. In addition, peptide 5 exhibited selective activity towards the pre-erythrocytic stage (98% of activity against P. gallinaceum), thus suggesting that it may be possible to design peptides that target specific stages of the malaria life cycle. The Ang II derived stable scaffolds presented here may provide the basis for development of a new generation of peptide-based drugs for the treatment of malaria.

Vasorelaxant activity of twenty-one physiologically relevant (poly)phenolic metabolites on isolated mouse arteries.

Polyphenols are beneficial for health, but are metabolised after consumption. We compared the vasorelaxant capacity of twenty-one physiologically relevant polyphenol metabolites in isolated mouse arteries. Hesperetin, urolithins and ferulic acid-4-O-sulfate - not their glucuronidated forms or ferulic acid - caused vasorelaxation. Therefore, we advise the use of relevant conjugates in future mechanistic research.

A critical role for cystathionine-ß-synthase in hydrogen sulfide-mediated hypoxic relaxation of the coronary artery.

Hypoxia-induced coronary artery vasodilatation protects the heart by increasing blood flow under ischemic conditions, however its mechanism is not fully elucidated. Hydrogen sulfide (H2S) is reported to be an oxygen sensor/transducer in the vasculature. The present study aimed to identify and characterise the role of H2S in the hypoxic response of the coronary artery, and to define the H2S synthetic enzymes involved. Immunoblotting and immunohistochemistry showed expression of all three H2S-producing enzymes, cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST), in porcine coronary artery. Artery segments were mounted for isometric tension recording; hypoxia caused a transient endothelium-dependent contraction followed by prolonged endothelium-independent relaxation. The CBS inhibitor amino-oxyacetate (AOAA) reduced both phases of the hypoxic response. The CSE inhibitor dl-propargylglycine (PPG) and aspartate (limits MPST) had no effect alone, but when applied together with AOAA the hypoxic relaxation response was further reduced. Exogenous H2S (Na2S and NaHS) produced concentration-dependent contraction followed by prolonged relaxation. Responses to both hypoxia and exogenous H2S were dependent on the endothelium, NO, cGMP, K+ channels and Cl-/HCO3- exchange. H2S production in coronary arteries was blocked by CBS inhibition (AOAA), but not by CSE inhibition (PPG). These data show that H2S is an endogenous mediator of the hypoxic response in coronary arteries. Of the three H2S-producing enzymes, CBS, expressed in the vascular smooth muscle, appears to be the most important for H2S generated during hypoxic relaxation of the coronary artery. A contribution from other H2S-producing enzymes only becomes apparent when CBS activity is inhibited.

Pregnancy Augments VEGF-Stimulated In Vitro Angiogenesis and Vasodilator (NO and H2S) Production in Human Uterine Artery Endothelial Cells.

Context: Augmented uterine artery (UA) production of vasodilators, including nitric oxide (NO) and hydrogen sulfide (H2S), has been implicated in pregnancy-associated and agonist-stimulated rise in uterine blood flow that is rate-limiting to pregnancy health. Objective: Developing a human UA endothelial cell (hUAEC) culture model from main UAs of nonpregnant (NP) and pregnant (P) women for testing a hypothesis that pregnancy augments endothelial NO and H2S production and endothelial reactivity to vascular endothelial growth factor (VEGF). Design: Main UAs from NP and P women were used for developing hUAEC culture models. Comparisons were made between NP- and P-hUAECs in in vitro angiogenesis, activation of cell signaling, expression of endothelial NO synthase (eNOS) and H2S-producing enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase, and NO/H2S production upon VEGF stimulation. Results: NP- and P-hUAECs displayed a typical cobblestone-like shape in culture and acetylated low-density lipoprotein uptake, stained positively for endothelial and negatively for smooth muscle markers, maintained key signaling proteins during passage, and had statistically significant greater eNOS and CBS proteins in P- vs NP-hUAECs. Treatment with VEGF stimulated in vitro angiogenesis and eNOS protein and NO production only in P-hUEACs and more robust cell signaling in P- vs NP-hUAECs. VEGF stimulated CBS protein expression, accounting for VEGF-stimulated H2S production in hUAECs. Conclusion: Comparisons between NP- and P-hUAECs reveal that pregnancy augments VEGF-stimulated in vitro angiogenesis and NO/H2S production in hUAECs, showing that the newly established hUAEC model provides a critical in vitro tool for understanding human uterine hemodynamics.

The role of eNOS on the compensatory regulation of vascular tonus by H2S in mouse carotid arteries.

The gasotransmitter nitric oxide (NO) has an important role in vascular function and a decrease in its bioavailability is accepted as a main pathological mechanism for cardiovascular diseases. However, other gasotransmitters such as hydrogen sulfide (H2S) are also generated by the endothelium and can also affect vascular tone and a crosstalk may exist between H2S and NO. We therefore investigated the consequences of deficiency, replacement or overexpression of endothelial nitric oxide synthase (eNOS) on H2S-induced vascular responses in murine carotid arteries. In pre-contracted carotid arteries from wild-type (WT) mice, l-cysteine elicited relaxation that was inhibited by the H2S synthesis inhibitor amino-oxyacetic acid (AOAA). Genetic deletion of eNOS increased l-cysteine-induced relaxation compared to WT, but the replacement of eNOS by adenoviral transfection or H2S synthesis inhibition by AOAA reversed it. Furthermore, eNOS deletion did not alter NaHS-induced relaxation in carotid arteries while eNOS overexpression/replacement increased NaHS-induced relaxation responses in carotid arteries from WT or eNOS-/-. We suggest that, endogenously produced H2S can compensate for impaired vasodilatory responses in the absence of NO to maintain vascular patency; while, eNOS abundance can limit endogenous H2S-induced vascular responses in mice carotid arteries. Our result suggests that endogenous vs. exogenous H2S-induced relaxation are reciprocally regulated by NO in mice carotid arteries.

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries.

Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N- (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid prostacyclin receptor (IP receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP receptor antagonists (CAY10441 and 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]-3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E receptor 3 antagonist enhanced the vasodilation induced by all IP receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP receptor agonists, works as a selective IP receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.

Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a Detoxifying Enzyme.

The enzyme xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism in the highest uricotelic primates. XOR is an enzyme with dehydrogenase activity that, in mammals, may be converted into oxidase activity under a variety of pathophysiologic conditions. XOR activity is highly regulated at the transcriptional and post-translational levels and may generate reactive oxygen and nitrogen species, which trigger different consequences, ranging from cytotoxicity to inflammation. The low specificity for substrates allows XOR to metabolize a number of endogenous metabolites and a variety of exogenous compounds, including drugs. The present review focuses on the role of XOR as a drug-metabolizing enzyme, specifically for drugs with anticancer, antimicrobial, antiviral, immunosuppressive or vasodilator activities, as well as drugs acting on metabolism or inducing XOR expression. XOR has an activating role that is essential to the pharmacological action of quinone drugs, cyadox, antiviral nucleoside analogues, allopurinol, nitrate and nitrite. XOR activity has a degradation function toward thiopurine nucleotides, pyrazinoic acid, methylxanthines and tolbutamide, whose half-life may be prolonged by the use of XOR inhibitors. In conclusion, to avoid potential drug interaction risks, such as a toxic excess of drug bioavailability or a loss of drug efficacy, caution is suggested in the use of XOR inhibitors, as in the case of hyperuricemic patients affected by gout or tumor lysis syndrome, when it is necessary to simultaneously administer therapeutic substances that are activated or degraded by the drug-metabolizing activity of XOR.

In vitro and in silico analysis of the vascular effects of asymmetrical N,N-bis(alkanol)amine aryl esters, novel multidrug resistance-reverting agents.

Asymmetrical N,N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca(2+) antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Cav1.2 channel current (I Ca1.2) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α1C subunit of the Cav1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1 µM (FRA77, GDE6, GDE19) and >10 µM (verapamil) in EA.hy926 cells, or >10 µM (FRA77, GDE6, GDE19) and at 100 µM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60 mM K(+) (K60)-induced contraction at concentrations ≥1 and ≥3 µM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10 µM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α1C subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited I Ca1.2 with IC50 values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers.

Medical Functions of Hydrogen Sulfide.

Hydrogen sulfide (H(2)S) is a gasomediator synthesized from L- and D-cysteine in various tissues. It is involved in a number of physiological and pathological processes. H(2)S exhibits antiatherosclerotic, vasodilator, and proangiogenic properties, and protects the kidney and heart from damage following ischemia/reperfusion injury. H(2)S donors may be natural or synthetic, and may be used for the safe treatment of a wide range of diseases. This review article summarizes the current state of knowledge of the therapeutic function of H(2)S.