Vascular Smooth Muscle Relaxation Research Articles

Effects of erythropoietin on spontaneous and oxytocin induced myometrial contractions in the nonpregnant rat.

Erythropoietin (EPO) is a glycoprotein hormone that regulates erythropoiesis. EPO activity has also been detected in a variety of tissue including the nervous system, and female and male reproductive organs. It has been shown that EPO causes relaxation in vascular smooth muscle. In the present study, we have investigated effects of EPO on spontaneous and oxytocin-induced contractions of non-pregnant rat myometrium. Myometrial stripes were obtained from adult Wistar rats at the oestrous stage. The samples were placed in an isolated organ chamber under physiological conditions and 1 g passive tension. Epoetin beta (rEPO) was added cumulatively at 0.1, 1 and 10 IU/ml concentrations to the myometrial samples showing regular spontaneous contractions for periods of 30 min. Frequency and amplitude of contractions were electrophysiologically recorded and analyzed by using a BIOPAC data acquisition system. rEPO inhibited both area under curve and frequency of spontaneous contractions (ANOVA, n1, 2 = 9, f1 = 20.938, f2 = 20.492, p1,2 = 0.000). The inhibitory effect was insignificant at 0.1 mIU/ml rEPO level (Tukey HSD, p1 = 0.051, p2 = 0.581). In the oxytocin treated myometrial samples, a single dose of 1 IU/ml rEPO was studied. The area under curve and frequency values of these samples were inhibited by rEPO (Student's t-test, n = 9, t1 = 4.776, p1 = 0.000; t2 = 2.835, p2 = 0.012, respectively). rEPO inhibited spontaneous and oxytocin-induced rat myometrial contractions at 1 and 10 IU/ml concentrations. It appeared that the effect was dose-dependent.

The AMP-Dependent Protein Kinase (AMPK) Activator A-769662 Causes Arterial Relaxation by Reducing Cytosolic Free Calcium Independently of an Increase in AMPK Phosphorylation.

Although recent studies reveal that activation of the metabolic and Ca2+ sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM) contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC). A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca2+- and GTPγS-induced contractions in β-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca2+ signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca2+]i), inhibited a stimulus-induced increase in [Ca2+]i, and inhibited a cyclopiazonic acid (CPA)-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca2+]i. Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca2+ mobilization pathway, possibly involving CPA-dependent Ca2+ entry via an AMPK-independent pathway. That MET activated AMPK without causing arterial relaxation suggests that AMPK activation does not necessarily cause VSM relaxation.

Fluorescent Probes and Selective Inhibitors for Biological Studies of Hydrogen Sulfide- and Polysulfide-Mediated Signaling.

Significance: Hydrogen sulfide (H2S) plays roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. Also, hydropersulfide (R−S−SH) and polysulfide (−S−Sn−S−) have recently been identified as reactive sulfur species (RSS) that regulate the bioactivities of multiple proteins via S-sulfhydration of cysteine residues (protein Cys−SSH) and show cytoprotection. Chemical tools such as fluorescent probes and selective inhibitors are needed to establish in detail the physiological roles of H2S and polysulfide.Recent Advances: Although many fluorescent probes for H2S are available, fluorescent probes for hydropersulfide and polysulfide have only recently been developed and used to detect these sulfur species in living cells.Critical Issues: In this review, we summarize recent progress in developing chemical tools for the study of H2S, hydropersulfide, and polysulfide, covering fluorescent probes based on various design strategies and selective inhibitors of H2S- and polysulfide-producing enzymes (cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase), and we summarize their applications in biological studies.Future Directions: Despite recent progress, the precise biological functions of H2S, hydropersulfide, and polysulfide remain to be fully established. Fluorescent probes and selective inhibitors are effective chemical tools to study the physiological roles of these sulfur molecules in living cells and tissues. Therefore, further development of a broad range of practical fluorescent probes and selective inhibitors as tools for studies of RSS biology is currently attracting great interest. Antioxid. Redox Signal. 27, 669–683.

Abstract P501: Editing of Myosin Phosphatase Gene as a Novel Approach for Vasodilator Sensitization and Lowering of Blood Pressure

Myosin Phosphatase (MP) is the primary effector of vascular smooth muscle (VSM) relaxation and a key end target of signaling pathways that regulate vessel tone. Regulated splicing of alternative Exon24 (E24) of Myosin Phosphatase Regulatory/ Targeting subunit (MYPT1) sets vasodilator sensitivity. Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGK1α binding and NO/cGMP activation of MP resulting in vasodilation. Inclusion of 31 nt E24 shifts the reading frame coding for a Mypt1 isoform with a distinct C-terminus (LZ-) that is unresponsive to NO/cGMP. We are using two editing approaches to test the function of Mypt1 E24 splice variants in the control of BP in vivo. First, LoxP sites were inserted in introns flanking E24, crossed with smMHCCre ER , and treated with Tamoxifen to achieve smooth muscle-specific cKO of E24 (SMcKO E24), thereby converting Mypt1 to the LZ+ isoform. E24 cKO mice had mean BP that was 15 + 3 mmHg lower than control (n=3-5; p<0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC 50 : 2.1+0.5 nM vs 18.2+5.6 μM; n=5-6, p<0.05). We now are developing CRISPR/CAS9 editing of Mypt1 for translation into humans with hypertension. Guide(g)RNAs targeting E24 were designed using Benchling.com and selected for further study based on predicted efficacy, specificity (>10%,>60%) and cross-species conservation. Plasmids were generated by sub-cloning of oligonucleotides into the parent pX601 plasmid for the purpose of co-expression of gRNA and saCas9. These plasmids were transfected into HEK293 cells singly and in combinations and Mypt1 gene editing assayed by PCR, Surveyor nuclease assays and sequencing of genomic DNA. Single gRNAs yielded deletions of 1-3 nt. Combinations yielded deletions of 104-334 nt that removed >80% of E24 with an efficiency of editing that varied from 10% (gRNAs 6+9 and 5+9) to 40% (gRNAs 6+11 and 5+11). We have now generated AAVgE24 and are testing their efficiency of editing of VSM in vivo. These studies support that AAV mediated CRISPR/Cas9 editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans.

Effect of nicorandil on QT dispersion in patients with stable angina pectoris undergoing elective angioplasty: A triple-blind, randomized, placebo-controlled study.

BackgroundNicorandil leads to the relaxation of fine vascular smooth muscle, and thus causes vasodilatation of major epicardial. Also, it has anti-arrhythmic and cardio-protective effects by improving reperfusion, and ultimately leads to a reduction in microvascular damage caused by percutaneous coronary intervention (PCI).ObjectiveThe aim of this study was to determine the effect of nicorandil on QT interval dispersion (QTd) in patients with stable angina pectoris during elective angioplasty.MethodsThis triple-blind and randomized clinical trial was performed on patients with stable angina pectoris, candidates for elective angiography referred to Imam Reza and Ghaem hospitals in Mashhad, Iran, between January and October 2016. The patients were randomly assigned to one of two groups receiving nicorandil (60 mg as 20 mg before and 40 mg after PCI) and placebo. All the patients underwent electrocardiography 12 hours before and 12 hours after PCI. The values of maximal corrected QT interval (QTc max) and QTd in these intervals, and the levels of changes in the QTd (QTd difference before angiography and after PCI) were compared between the two groups. Data were analyzed statistically using SPSS version 18 software via Chi-square and Independent-samples t-test.ResultsThis study was performed on 90 patients (55 males and 35 females) with a mean age of 58.6±10.8 years, on two groups of 45 people. The two groups were matched for age, body mass index, cardiovascular risk factors and baseline testing. The QTd before angiography had no statistically significant difference between the patients of both groups (control: 77.7±17.1 vs. nicorandil: 80.7±14.2 ms; p=0.371). The QTd after PCI in the nicorandil group was lower than the control group (48.1±14.2 vs. 59.2±15.6 ms; p=0.000). The decrease rate in QTd had a statistically significant difference between the two groups (control: 18.9±11.0 vs. nicorandil: 33.5±9.5 ms; p=0.000).ConclusionsThe results of this study showed that oral administration of nicorandil around the PCI could further reduce QTd following PCI, compared to the control group.Trial registrationThe trial was registered at the Iranian Registry of Clinical Trials (http://www.irct.ir) with the Irct ID: IRCT2016120631159N1FundingThe authors received no financial support for the research, authorship, and/or publication of this article.

Calcium/calmodulin-dependent kinase 2 mediates Epac-induced spontaneous transient outward currents in rat vascular smooth muscle.

Key points The Ca2+ and redox‐sensing enzyme Ca2+/calmodulin‐dependent kinase 2 (CaMKII) is a crucial and well‐established signalling molecule in the heart and brain.In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII.The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent Ca2+ influx. How Epac activates STOCs is unknown.In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII.To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)‐sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+‐activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage‐dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′, 5‐cyclic monophosphate‐AM (8‐pCPT‐AM) induces autophosphorylation (activation) of calcium/calmodulin‐dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8‐pCPT‐AM‐induced increases in STOC activity. Epac‐induced CaMKII activation is probably initiated by inositol 1,4,5‐trisphosphate (IP3)‐mobilized Ca2+: 8‐pCPT‐AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8‐pCPT‐AM‐mediated CaMKII phosphorylation and STOC activity. 8‐pCPT‐AM does not directly activate BKCa channels, but STOCs cannot be generated by 8‐pCPT‐AM in the presence of ryanodine. Furthermore, exposure to 8‐pCPT‐AM significantly slows the initial rate of [Ca2+]i rise induced by the RyR activator caffeine without significantly affecting the caffeine‐induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac‐mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox‐sensing enzyme in the regulation of vascular tone and blood flow.

H2S confers colonoprotection against TNBS-induced colitis by HO-1 upregulation in rats.

Hydrogen sulfide (H2S) is an endogenous mediator that contributes to many important physiological processes including vasodilation and vascular smooth muscle relaxation; in turn, preventing tissue damage and reducing inflammation. Heme oxygenase (HO) enzymes, of which HO-1 is inducible by harmful stimuli, were found to regulate intestinal inflammation in experimental animal models of colitis. We aimed to investigate the protective effects of H2S against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats, and whether HO enzyme system is involved in the H2S-induced colonic cytoprotection. Male Wistar rats were treated with TNBS to induce colitis, and H2S donor (Lawesson's reagent) was prepared two times/day at different concentrations, and delivered per os (from day 1 to day 3). Our results suggest that daily treatment (2 times/day) with H2S donor, could significantly decrease the extent of colonic inflammation compared to vehicle treatment, and the most effective daily dose of H2S donor against inflammation was 18.75µM/kg/day. Per os administration of H2S donor increased the colonic HO enzyme activity; on the contrary, the protective effect of H2S was abolished by the co-treatment with HO inhibitor. Our findings suggest that H2S confers colonoprotection, probably by modulation of anti-inflammatory parameters and HO enzyme activity.

Contribution of Rho-kinase and Adenosine Monophosphate-Activated Protein Kinase Signaling Pathways to Endothelium-Derived Contracting Factors Responses.

Vascular tonus is controlled by endothelium-derived relaxing factor (EDRF), endothelium-derived hyperpolarizing factor (EDHF) and endothelium-derived contracting factor (EDCF) under physiological circumstances. In pathological conditions, impairment of endothelium-derived relaxation can be caused by both decrease in EDRF release and increase in EDCF release. The increase in EDCF is observed with diseases such as hypertension and diabetes. The contribution of Rho-kinase and activated protein kinase (AMPK), which have opposite effects, to the increased EDCF responses was investigated. Rho-kinases are the effectors of Rho which is one of the small guanosine triphosphate-binding proteins. They increase cytosolic Ca+2 concentration and cause vascular smooth muscle to contract, keeping myosin light chain (MLC) in phosphorylated state by affecting myosin phosphatase target subunit which dephosphorylates the MLC. The activities of Rho-kinases increase with the increase of EDCF function. AMPK is the energy sensor of the cell. It provides a vasculoprotective effect by causing endothelium-dependent and endothelium-independent relaxation in smooth muscle. In contrast to Rho-kinase pathway activity, AMPK pathway activity decreases with diseases in which the EDCF function increases. In cases such as diabetes and hypertension that endothelial function impairs toward vasocontraction, it is considered that evaluating Rho-kinase and AMPK pathways which mediate contraction and relaxation in vascular smooth muscle respectively, would provide clues on choosing therapeutic target for pathologies in which endothelial dysfunction is observed.

Triggered and Tunable Hydrogen Sulfide Release from Photogenerated Thiobenzaldehydes.

Hydrogen sulfide (H2 S) has been identified as an important cell-signaling mediator and has a number of biological functions, such as vascular smooth muscle relaxation, neurotransmission, and regulation of inflammation. A facile and versatile approach for H2 S production initiated by light irradiation and controlled by reaction with an amine or an amino acid was developed. The donor was synthesized in a one-pot reaction, and simple crystallization led to a yield of approximately 90 %. The synthetic strategy is scalable and versatile, and the H2 S donors can be expressed ina number of different molecular and macromolecular forms, including crystalline small-molecule compounds, water-soluble polymers, polystyrene films, and hydrogels. The H2 S donors based on polystyrene film and hydrogel were used as cell-culture scaffolds. The H2 S donor based on water-soluble polymer was applied in photocontrolled inhibition of P-selectin expression on human platelets and subsequent regulation of platelet aggregation. This study provides the simplest controllable H2 S source to study its biological functions. The developed materials are also new therapeutic platforms to deliver H2 S, as there is no accumulation of toxic byproducts, and the donor materials from polystyrene films and hydrogels can be readily removed after releasing H2 S.

Chantal M. Boulanger: Exploring the Endothelium.

The scientific evolution of Chantal M. Boulanger, PharmD, PhD, has taken her from a French village’s one-room schoolhouse to the Mayo Clinic to a leadership position at the Institut National de la Sante et de la Recherche Medicale (Inserm) in Paris. Chantal M. Boulanger While completing pharmacy studies at the Universite de Lorraine, Boulanger leapt at the chance to spend summers in research laboratories with the aim of pursuing a PhD. Her molecular pharmacology doctoral work, on the intracellular mechanisms associated with vascular smooth muscle relaxation, was conducted at the Universite de Strasbourg in the program headed by Jean-Claude Stoclet. Boulanger’s research career has focused on the role of endothelial cells as a key player in vascular disease development and as a target of cardiovascular risk factors. While she was a research associate at the University Hospital of Basel, Switzerland, Boulanger and T.F. Luscher demonstrated that production of the vasoconstrictor endothelin is downregulated by nitric oxide1 and stimulated by oxidized lipids2 in isolated endothelium, findings that were later confirmed in intact animals and provided new pharmacological targets to address endothelial dysfunction. In a change that required a major shift in laboratory techniques and models, since 2000 Boulanger’s research team at Inserm has investigated the extracellular microvesicles produced by endothelial cell activation. The team demonstrated that circulating microvesicles from patients with myocardial infarction triggered endothelial dysfunction, even in angiographically normal arteries.3 They identified microvesicles in human atherosclerotic plaque as potent prothrombogenic mediators that might represent a major determinant of plaque neovascularization and vulnerability.4 Later, they showed that circulating endothelial microvesicles were associated with cardiovascular and metabolic risk factors in participants in the Framingham offspring study who had no history of cardiovascular disease.5 Currently, Boulanger’s research addresses the regulation of micro-RNA packaging in extracellular vesicles and resulting …

Methylnicotinate stimulated prostaglandin synthesis in patients with schizophrenia: A preliminary investigation

Schizophrenia is a serious mental illness of unclear aetiology. The reduced ability of methylnicotinate to induce a topical vasodilatory response in patients with the disorder is well established. Methylnicotinate causes vasodilation via stimulating the release of prostaglandins (including prostaglandin D2) in the skin which in turn leads to relaxation of vascular smooth muscle. To determine whether the abnormality is likely to be due to decreased prostaglandin production, or a decreased effect of prostaglandins upon the vessels, topical methylnicotinate was applied to the forearms of patients with schizophrenia or healthy controls, followed by rating of the resulting erythema. The concentration of prostaglandin D2 and its metabolite 11β-prostaglandin F2α in the blood draining the arm was also measured. Although erythema was reduced in the patient group, this was not correlated with plasma prostaglandin concentrations. This data suggests the abnormality underlying the reduced potency of methylnicotinate to produce vasodilation in the disorder occurs downstream of prostaglandin synthesis possibly within the vasculature itself.

Mechanism of resveratrol-induced relaxation in the human gallbladder

BackgroundResveratrol is a polyphenolic compound extracted from plants and is also a constituent of red wine. Resveratrol produces relaxation of vascular smooth muscle and may prevent cardiovascular diseases. Although resveratrol has been reported to cause relaxation of the guinea pig gallbladder, limited data are available about the effect of resveratrol on the gallbladder smooth muscle in humans. The purpose of this study was to investigate the relaxation effects of resveratrol in human gallbladder muscle strips.MethodsWe studied the relaxant effects of resveratrol in human gallbladder. In addition, we also investigated mechanism of resveratrol-induced relaxation in human gallbladder by tetraethylammonium (a non-selective potassium channels blocker), iberiotoxin (an inhibitor of large conductance calcium-activated potassium channel), glibenclamide (an ATP-sensitive potassium channel blocker), charybdotoxin (an inhibitor of large conductance calcium-activated potassium channels and slowly inactivating voltage-gated potassium channels), apamine (a selective inhibitor of the small conductance calcium-activated potassium channel), KT 5720 (a cAMP-dependent protein kinase A inhibitor), KT 5823 (a cGMP-dependent protein kinase G inhibitor), NG-Nitro-L-arginine (a competitive inhibitor of nitric oxide synthase), tetrodotoxin (a selective neuronal Na+ channel blocker), and ω-conotoxin GVIA (a selective neuronal Ca2+ channel blocker).ResultsThe present study showed that resveratrol has relaxant effects in human gallbladder muscle strips. In addition, we found that resveratrol-induced relaxation in human gallbladder is associated with nitric oxide, ATP-sensitive potassium channel, and large conductance calcium-activated potassium channel pathways.ConclusionsThis study provides the first evidence concerning the relaxant effects of resveratrol in human gallbladder muscle strips. Furthermore, these results demonstrate that resveratrol is a potential new drug or health supplement in the treatment of biliary colic.

Cardioprotective function of progesterone: A new perspective

Cardioprotective mechanism in females during reproductive phase is unclear. New perspective explains cardioprotective function of progesterone. Progesterone, the intermediary metabolic product in synthesis of steroid hormones, with t/2 of 30 min, stimulates respiratory center inducing respiratory alkalosis in turn lowers plasma ionic calcium. This results in decreased blood coagulability and cardiac contractility along with smooth muscle relaxation. Vascular smooth muscle relaxation, leading to generalized vasodilatation causes better tissue perfusion resulting in diminished erythropoietin, and erythrocyte count. This reduces blood viscosity and afterload on the heart. Cardiac contractility remains balanced due to opposing influences of decreased plasma ionic calcium and raised basal body temperature. On sudden withdrawal of progesterone, as during premenstrual, postpartum and postmenopausal periods, cardioprotective changes are reversed. The awareness helps to build better body buffer system, aiming to correct acid-base imbalance, during expected fluctuations of progesterone.

Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.

Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.

Cold-induced sympathetic tone modifies the impact of endothelium-dependent vasodilation in the finger pulp.

In thermoneutral and cold subjects, the sympathetic nervous system regulates skin blood flow by adjusting frequency of the tonic vasoconstrictor impulses. However, the way these thermoregulatory impulses influence the vascular endothelium is not well known. We studied how the sympathetic nervous system influences endothelium-dependent vasodilation (EDV) caused by shear stress in skin containing arteriovenous anastomoses (AVAs) and arterioles in healthy subjects. Thirteen healthy subjects were exposed to thermoneutral (29°C) and cold (22°C) ambient temperatures on separate days. EDV was induced by releasing suprasystolic pressure cuff applied to the forearm or third finger after 4min. Bilateral laser Doppler flux from the finger pulp, dorsal finger and dorsal wrist was measured together with ultrasound Doppler from the right radial artery. Absolute EDV response (EDV peak minus baseline) and normalized relative EDV response (ratio EDV peak/baseline) were calculated (median, 95% confidence interval). The relative EDV response reflect the size of EDV response independent of the baseline level and is thus used to compare the EDV responses in the finger pulp and wrist skin in the two temperature conditions. In finger pulp (dominated by AVAs), the absolute EDV response (flux, au) in thermoneutral (137.8 (67.5, 168.8)) and cold (130.3 (97.2, 154.9)) was the same (p=0.85), whereas the relative EDV response was significantly higher in cold (3.6 (2.5, 5.9)) than in thermoneutral (1.4 (1.1, 1.6), p=0.002). The same patterns were found in the radial artery. In the dorsal wrist (dominated by arterioles) the absolute EDV response (flux, au) was smaller in cold (30.9 (15.91, 38.0)) than in thermoneutral (52.1 (38.4, 57.8), p=0.04), whereas the relative EDV responses in cold (3.5 (2.3, 4.2)), and thermoneutral (2.3 (1.6, 2.7)) were equal (p=0.16). The relative EDV responses show that the impact of EDV on skin perfusion in cold conditions is significantly greater in the finger pulp than in wrist skin. However, the absolute EDV responses indicate that vascular smooth muscle relaxation during EDV is probably not affected by higher mild cold-induced sympathetic activity either in AVAs or in arterioles.

MiR-153/Kv7.4: a novel molecular axis in the regulation of hypertension.

This editorial refers to ‘MicroRNA-153 targeting of KCNQ4 contributes to vascular dysfunction in hypertension’, by G. Carr et al. , pp. 581–589 Characterized by a steady increase in blood pressure values, hypertension is a complex, multifactorial disease resulting from a combination of environmental and genetic factors. Blood pressure is a function of peripheral resistance, which in turn depends on arterial diameter. Several molecular mechanisms have been described as regulators of arterial diameter both chronically, leading to vascular remodelling, and acutely, by contraction or relaxation of vascular smooth muscle in the vessel wall. Although decades of research proposed several advances in anti-hypertensive therapies, there are still patients with uncontrolled hypertension, thus pressing the search for novel mechanisms underlying hypertension and its related cardiovascular modifications. MicroRNAs, small non-coding RNAs that target messenger RNAs at the post-transcriptional level, are involved in virtually all biological processes, including cellular proliferation, apoptosis, and differentiation.1 Deregulation of many individual microRNAs has been linked to the development and progression of cardiovascular diseases, …

Intestinal and vascular smooth muscle relaxant effect of Viscum album explains its medicinal use in hyperactive gut disorders and hypertension.

BackgroundViscum album has shown inhibitory effect on different smooth muscles but underlying mechanisms in gut and vascular smooth muscles are not well defined. Additionally, the plant has also importance in managing hyperactive gut and cardiovascular disorders. The current study was aimed to probe a pharmacological base of the smooth muscle relaxant effect of V. album in gut and vascular preparations.MethodsV. album crude extract (Va. Cr) and its ethyl acetate fraction (Va. EtAc) were studied using in vitro techniques. The antispasmodic activity was performed using isolated rabbit jejunum while the vasorelaxant effects were studied in rabbit aortic rings.ResultsVa. Cr and Va. EtAc inhibited spontaneous and high K+-induced contractions with EC50 values of 0.31 mg/mL (0.15–0.57) and 0.62 mg/mL (0.3–0.95), respectively. This advocates an antispasmodic effect probably operated through calcium channels blockade (CBB). The proposed mechanism was confirmed by a pretreatment of the tissue with Va. Cr (0.01–0.3 mg/mL), which shifted the Ca++ concentration-response curves (CRCs) rightward, similar to verapamil. Moreover, Va. Cr showed a partial relaxation against high K+ and PE (1 μM) induced contractions in isolated rabbit aorta rings. Va. EtAc caused complete relaxation of high K+ precontraction and partially relaxed PE (1 μM) induced contractions, suggesting inhibitory effect on Ca++ entry, in addition to other possible mechanisms. CRCs were shifted to the right correspondingly to verapamil when the aortic rings were pretreated with Va. Cr and Va. EtAc.ConclusionsThese data indicated that Va. Cr possesses smooth muscle relaxant effect mediated through voltage-dependent Ca++ channel blockade (CCB), which explains its spasmolytic and vasorelaxant activity. The CCB activity is concentrated more in Va. EtAc. This study provides an evidence for the medicinal importance of V. album in gut spasm and possibly hypertension.

Screening and identification of Caulis Sinomenii bioactive ingredients with dual-target NF-κB inhibition and β2- AR agonizing activities.

Caulis Sinomenii (CS) is a valuable traditional medicine in China. Its extract can act as an anti-inflammatory agent and a vascular smooth muscle relaxant. However, the underlying mechanisms remain unknown. In this study, we developed a simple dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with a dual-target bioactive screening assay for anti-inflammatory and antispasmodic activities to characterize the chemical structure of various bioactive compounds of CS rapidly. Seven potential NF-κB inhibitors were identified, including laudanosoline-1-O-xylopyranose, 6-O-methyl-laudanosoline-1-O-glucopyranoside, menisperine, sinomenine, laurifoline, magnoflorine and norsinoacutin. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which laudanosoline-1-O-d-xylopyranose and menisperine were revealed as novel NF-κB inhibitors. Among the seven identified alkaloids, three potential β2 -adrenergic receptor agonists, including sinomenine, magnoflorine and laurifoline, were characterized using a luciferase reporter system to measure for the activity of β2 -adrenergic receptor agonists. Finally, sinomenine, magnoflorine and laurifoline were identified not only as potential NF-κB inhibitors but also as potential β2 -adrenegic receptor agonists, which is the first time this has been reported. Molecular dynamic simulation and docking results suggest that the three dual-bioactive constituents could not only inhibit Pseudomonas aeruginosa PAK strain-induced inflammatory responses via a negative regulation of the Braf protein that participates in MAPK signaling pathway but also activate the β2 -adrenegic receptor. These results suggest that CS extract has dual signaling activities with potential clinical application as a novel drug for asthma.

Role of Mitochondria in Cerebral Vascular Function: Energy Production, Cellular Protection, and Regulation of Vascular Tone.

Mitochondria not only produce energy in the form of ATP to support the activities of cells comprising the neurovascular unit, but mitochondrial events, such as depolarization and/or ROS release, also initiate signaling events which protect the endothelium and neurons against lethal stresses via pre-/postconditioning as well as promote changes in cerebral vascular tone. Mitochondrial depolarization in vascular smooth muscle (VSM), via pharmacological activation of the ATP-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels), leads to vasorelaxation through generation of calcium sparks by the sarcoplasmic reticulum and subsequent downstream signaling mechanisms. Increased release of ROS by mitochondria has similar effects. Relaxation of VSM can also be indirectly achieved via actions of nitric oxide (NO) and other vasoactive agents produced by endothelium, perivascular and parenchymal nerves, and astroglia following mitochondrial activation. Additionally, NO production following mitochondrial activation is involved in neuronal preconditioning. Cerebral arteries from female rats have greater mitochondrial mass and respiration and enhanced cerebral arterial dilation to mitochondrial activators. Preexisting chronic conditions such as insulin resistance and/or diabetes impair mitoKATP channel relaxation of cerebral arteries and preconditioning. Surprisingly, mitoKATP channel function after transient ischemia appears to be retained in the endothelium of large cerebral arteries despite generalized cerebral vascular dysfunction. Thus, mitochondrial mechanisms may represent the elusive signaling link between metabolic rate and blood flow as well as mediators of vascular change according to physiological status. Mitochondrial mechanisms are an important, but underutilized target for improving vascular function and decreasing brain injury in stroke patients. © 2016 American Physiological Society. Compr Physiol 6:1529-1548, 2016.

Unbiased Profile of MicroRNA Expression in Ascending Aortic Aneurysm Tissue Appoints Molecular Pathways Contributing to the Pathology

Complex etiopathogenesis of ascending aortic aneurysm suggests contribution of epigenetic mechanisms in its development. Several studies appointed microRNAs (miRs) as essential epigenetic factors in various human diseases; however, little is known about their role in ascending aortic aneurysm. Therefore, the aim of this study was to perform unbiased molecular screening of miRs expression in aneurysmal tissue and establish their functions on a transcriptional level. Samples of ascending aortic tissue were obtained from 15 patients, and total RNA was isolated separately from aneurysmal and unaffected aortic tissue obtained from the same patient. Expression of the complete panel of human miRs was assessed by quantitative real-time polymerase chain reaction. Using bioinformatic tools, 13 genes were selected that were putatively regulated by overexpressed miRs. Expression level of transcripts were evaluated by quantitative real-time polymerase chain reaction and correlated with their targeting miRs. Overexpression of 10 miRs distinguished aneurysmal tissue from the unchanged one. These miRs were involved in cell senescence (miR-191-5p), maintenance of vascular integrity (miR-126-3p and miR-374-5p), nitric oxide-dependent vascular relaxation (miR-21-5p), smooth muscle differentiation, and contractility (miR-145- 3p, miR-29c-3p, miR-133a-3p, miR-186-5p, miR-143-3p, and miR-24-3p), and correlated with abundance of its miR targets. Altered expression of particular miRs selectively in the affected tissue indicate their role as factors that trigger pathways of aneurysmal transformation. Limited reparative properties due to overexpression of miR-191 may play a crucial role for aneurysm enlargement, whereas nitric oxide-dependent relaxation of vascular smooth muscle mediated by miR-21 offers an attractive explanation of the aneurysm's initiation, and is confirmed in experimental conditions.