Contraction Of Aortic Rings Research Articles

Abstract P182: The Novel Perivascular Adipose Tissue Adipokine, Chemerin, Signals Through G i - and Calcium-Dependent Mechanisms

Chemerin is an adipokine associated with inflammation, increased blood pressure, and may be a link between the pathologies of obesity and hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via the chemerin receptor and calcium flux in smooth muscle cells. Known mediators of the amplified arterial responsiveness seen in hypertension (L-type Ca 2+ channels, Src, and Rho kinase) were interrogated by isometric contraction of rat aortic rings in parallel with calcium kinetics of rat aortic smooth muscle cells. Western blots were also used to observe phosphorylation of Erk/MAPK. Chemerin-9 (nonapeptide of the chemerin S 157 isoform) caused a concentration-dependent contraction of isolated aorta (EC 50 100 nM) and elicited a concentration-dependent intracellular calcium response (EC 50 10 nM). Both calcium influx and isometric contraction, respectively, were reduced (units of “% of vehicle response”) by Pertussis toxin (G i inhibitor; 0±3% and 23±9%), verapamil (L-type Ca 2+ channel inhibitor; 38±20% and 23±4%), PP1 (Src inhibitor; 43±23% and 15±4%), and Y27632 (Rho Kinase inhibitor; 58±23% and 22±4%) but U73122 (PLC inhibitor) had little to no effect (71±31% and 71±12%). PD098059 (Erk/MAPK inhibitor) did not inhibit chemerin-9 induced contraction (117±19%) and phosphorylation did not change after chemerin-9 stimulation [1.12±0.14 (44 kDa) and 1.11±0.29 (42 kDa) fold-increase with chemerin-9 contraction compared to vehicle, p>0.05]. The chemerin receptor-selective antagonist CCX832 inhibited chemerin-9-induced calcium flux and aortic contraction and calcium flux (0.1±10.3% and 10±7%). These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through the G i -linked chemerin receptor to activate L-type Ca 2+ channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us one step closer to targeting chemerin as a unique form of therapy.

PS 10-04 HYPOTENSIVE EFFECT OF LOSARTAN THROUGH SUPPRESSING LEUKEMIA-ASSOCIATED RhoGEF EXPRESSION IN VASCULAR SMOOTH MUSCLE OF SPONTANEOUSLY HYPERTENSIVE RATS

Objective: To elucidate the mechanism that chronic blood pressure (BP) reduction caused by losartan is through the suppression of leukemia-associated Rho guanine nucleotide exchange factors (LARG) expression in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs). Design and Method: Four-week-old Wistar-Kyoto (WKY) rats and SHRs were treated with Losartan (20 mg/kg/day) for 8 weeks. BP was measured by the tail-cuff method. At 12 weeks, isometric contraction of rat aortic rings was measured with a force transducer and recorder. The target Rho guanine nucleotide exchange factors (RhoGEFs) messenger RNA (mRNA) and protein levels, and the myosin phosphatase target subunit 1 (MYPT-1) phosphorylation extent in aorta were determined using quantitative real-time polymerase chain reaction (qPCR) assay and Western blot analysis, respectively. Results: BP of four-week-old SHRs did not differ from that of age-matched WKY rats, whereas BP increased in twelve-week-old SHRs. Losartan treatment reduced BP in both rats’ strains, with greater extent in SHRs. The contractile force of aortic rings from SHRs treated with losartan was significantly lower than that from non-treated SHRs in response to angiotensin II. LARG protein expression was significantly higher in non-treated SHRs compared to WKY rats. After losartan treatment, both LARG protein level and phosphorylation extent of MYPT-1 were more suppressed in SHRs than in WKY rats. Conclusions: The study shows that chronic reduction of BP by losartan in SHRs is through suppressing LARG expression and MYPT-1 phosphorylation of VSMCs. Administration of losartan at early age of SHRs may help prevent hypertension development.

Increased vascular eNOS and cystathionine-γ-lyase protein after 6weeks oral administration of 3, 5, 7, 3', 4'-pentamethoxyflavone to middle-aged male rats.

Effects of treatment of middle-aged male rats with 3, 5, 7, 3', 4'-pentamethoxyflavone (PMF) on vascular and perivascular adipose tissue (PVAT) functions and blood chemistry were investigated. Rats received PMF (22mg/kg), orally or vehicle, twice a day for 6weeks. The PMF-treated rats had lower serum glucose, higher HDL-C levels, but no change in other parameters. Thoracic aortic and mesenteric rings of PMF treated rats produced lower maximal contraction to phenylephrine that was normalized by NG-nitro-L-arginine (L-NA) or endothelial removal. The aortic- and mesenteric rings of the PMF treated rats showed improved relaxation to acetylcholine, but not to glyceryl trinitrate, and had higher eNOS protein. DL-propargylglycine (PAG) caused greater increase in the baseline tension of the PMF-treated aortic ring and higher contraction to low concentrations of phenylephrine. PVAT lowered the contractile response of the L-NA pretreated aortic rings to phenylephrine for both groups, but PAG had no effect. The cystathionine-γ-lyase (CSE) protein of the thoracic rings, but not of the PVAT, shows increased expression after PMF treatment. Overall, PMF treatment of middle aged rats appeared to increase production of NO and H2S from the blood vessels by upregulating the expression of eNOS and CSE. PMF also decreased fasting serum glucose and increased HDL-C levels, with no toxicity to liver and kidney functions. Thus, PMF is a novel compound for possible use as a health product to prevent and/or to reduce the development of diabetes type II and/or cardiovascular disease.

Nitric oxide production contributes to Bacillus anthracis edema toxin-associated arterial hypotension and lethality: ex vivo and in vivo studies in the rat.

We showed previously that Bacillus anthracis edema toxin (ET), comprised of protective antigen (PA) and edema factor (EF), inhibits phenylephrine (PE)-induced contraction in rat aortic rings and these effects are diminished in endothelial-denuded rings. Therefore, employing rat aortic ring and in vivo models, we tested the hypothesis that nitric oxide (NO) contributes to ET's arterial effects. Compared with rings challenged with PA alone, ET (PA + EF) reduced PE-stimulated maximal contractile force (MCF) and increased the PE concentration producing 50% MCF (EC50) (P < 0.0001). Compared with placebo, l-nitro-arginine methyl-ester (l-NAME), an NO synthase (NOS) inhibitor, reduced ET's effects on MCF and EC50 in patterns that approached or were significant (P = 0.06 and 0.03, respectively). In animals challenged with 24-h ET infusions, l-NAME (0.5 or 1.0 mg·kg(-1)·h(-1)) coadministration increased survival to 17 of 28 animals (60.7%) compared with 4 of 27 (14.8%) given placebo (P = 0.01). Animals receiving l-NAME but no ET all survived. Compared with PBS challenge, ET increased NO levels at 24 h and l-NAME decreased these increases (P < 0.0001). ET infusion decreased mean arterial blood pressure (MAP) in placebo and l-NAME-treated animals (P < 0.0001) but l-NAME reduced decreases in MAP with ET from 9 to 24 h (P = 0.03 for the time interaction). S-methyl-l-thiocitrulline, a selective neuronal NOS inhibitor, had effects in rings and, at a high dose in vivo models, comparable to l-NAME, whereas N'-[3-(aminomethyl)benzyl]-acetimidamide, a selective inducible NOS inhibitor, did not. NO production contributes to ET's arterial relaxant, hypotensive, and lethal effects in the rat.

In vitro vasorelaxation mechanisms of Isoapiole extracted from Lemonfragrant Angelica Root on rat thoracic aorta

Ethnopharmcological relevanceLemonfragrant Angelica (Ostericum citriodorum (Hance) C. Q. Yuan & Shan) is a traditional Chinese herb for treatment of angina pectoris, stomach pain and abdominal pain. However, its active components and mechanisms of action were not well understood. Aims of the studyIn this study, we investigated whether the isoapiole extracted from Lemonfragrant Angelica Root (LAR) could directly stimulate the production of nitric oxide (NO) in vascular endothelial cells (VECs) and lead to the vascular relaxation Materials and methodsVascular activity experiments were performed in aortic rings isolated from Wistar rats using standard muscle bath procedures. Isoapiole was added with different concentrations (0.75, 2.5, 5μg/mL), and vessel relaxation of rat aortic rings pre-contracted with norepinephrine (NE) or potassium chloride was recorded. NO release from aortic rings exposed to isoapiole (5μg/mL) was measured by Griess method. The endothelial nitric oxide synthase (eNOS) expression in primary human umbilical vein endothelial cells (HUVECs) incubated with isoapiole was determined using Western blot and microplate reader assay. Classical receptor antagonists, channel and enzymatic inhibitors were used to check the mechanisms involved. ResultsIsoapiole (0.75, 2.5, 5μg/mL) inhibited norepinephrine-induced contraction in endothelium-intact rat aortic rings. However, a very weak relaxation of aortic rings was obtained in endothelium-denuded preparations. Isoapiole (0.75, 2.5, 5μg/mL) did not have vascular relaxative effect on neither endothelium-intact nor endothelium-denuded aortas pre-contracted with KCl (60mmol/L). The vasorelaxation effect of isoapiole on rat aortic rings was attenuated by the eNOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). This result suggested that suggested that the isoapiole action was at least partially mediated by promoting eNOS expression. It was further found that isoapiole (5μg/mL) increased NO production in isolated rat thoracic aorta rings. Isoapiole increased eNOS expression leading to NO production in HUVECs. ConclusionIsoapiole stimulates NO production in the endothelium, leading to vascular dilatation.

DL0805-2, a novel indazole derivative, relaxes angiotensin II-induced contractions of rat aortic rings by inhibiting Rho kinase and calcium fluxes.

DL0805-2 [N-(1H-indazol-5-yl)-1-(4-methylbenzyl) pyrrolidine-3-carboxamide] is a DL0805 derivative with more potent vasorelaxant activity and lower toxicity. This study was conducted to investigate the vasorelaxant mechanisms of DL0805-2 on angiotensin II (Ang II)-induced contractions of rat thoracic aortic rings in vitro. Rat thoracic aortic rings and rat aortic vascular smooth muscle cells (VSMCs) were pretreated with DL0805-2, and then stimulated with Ang II. The tension of the aortic rings was measured through an isometric force transducer. Ang II-induced protein phosphorylation, ROS production and F-actin formation were assessed with Western blotting and immunofluorescence assays. Intracellular free Ca(2+) concentrations were detected with Fluo-3 AM. Pretreatment with DL0805-2 (1-100 μmol/L) dose-dependently inhibited the constrictions of the aortic rings induced by a single dose of Ang II (10(-7) mol/L) or accumulative addition of Ang II (10(-10)-10(-7) mol/L). The vasodilatory effect of DL0805-2 was independent of endothelium. In the aortic rings, pretreatment with DL0805-2 (1, 3, and 10 μmol/L) suppressed Ang II-induced Ca(2+) influx and intracellular Ca(2+) mobilization, and Ang II-induced phosphorylation of two substrates of Rho kinase (MLC and MYPT1). In VSMCs, pretreatment with DL0805-2 (1, 3, and 10 μmol/L) also suppressed Ang II-induced Ca(2+) fluxes and phosphorylation of MLC and MYPT1. In addition, pretreatment with DL0805-2 attenuated ROS production and F-actin formation in the cells. DL0805-2 exerts a vasodilatory action in rat aortic rings through inhibiting the Rho/ROCK pathway and calcium fluxes.

The effect of urapidil, an alpha-1 adrenoceptor antagonist and a 5-HT1A agonist, on the vascular tone of the porcine coronary and pulmonary arteries, the rat aorta and the human pulmonary artery

Urapidil (Eupressyl®) an antihypertensive drug acting as an α1 antagonist and a 5-HT1A agonist, may be of special interest in the treatment of hypertension associated with preeclamptic toxaemia and hypoxia-induced pulmonary arterial vasoconstriction. However, the effect of urapidil on vascular tone has been poorly investigated. Vascular reactivity was evaluated using pulmonary and coronary arteries from 36 pigs, aortae from 22 rats and 9 human pulmonary artery samples suspended in organ chambers. Concentration-relaxation curves either to urapidil, 5-HT, or the 5-HT1A receptor agonist 8-OH-DPAT were constructed after pre-contraction of rings. Pig pulmonary and coronary artery rings were contracted with U46619, a thromboxane mimetic, rat aortic rings with either endothelin-1 or phenylephrine, and human pulmonary artery rings with U46619 or phenylephrine. Urapidil markedly inhibited phenylephrine-induced contractions in rat aortic rings with and without endothelium with a more pronounced effect observed in rings without endothelium. Both 5-HT and 8-OH-DPAT failed to induce relaxation in rat aortic rings with an intact endothelium. 5-HT, but not urapidil and 8-OH-DPAT, induced a concentration-dependent relaxation in the porcine coronary and pulmonary artery rings with an intact endothelium (P<0.05). 5-HT and phenylephrine but not urapidil caused concentration-dependent contractions in human pulmonary artery rings. The present findings, while confirming that urapidil is a potent inhibitor of α1-adrenoceptor-induced contraction, do not support the role of 5-HT1A receptor activation in the control of the vascular tone of the different types of arteries tested in response to urapidil. In addition, they indicate that urapidil seems to preferentially target arteries with endothelial dysfunction.

Heartwood extract of Rhus verniciflua Stokes and its active constituent fisetin attenuate vasoconstriction through calcium-dependent mechanism in rat aorta.

Rhus verniciflua Stokes (RVS) exert cardiovascular protective activity by promoting blood circulation, but its active ingredients and underlying mechanism have yet to be identified. This study investigated the vascular effects of RVS, focusing on vasoconstriction and smooth muscle Ca(2+) signaling. RVS heartwood extract attenuated contraction of aortic rings induced by the vasoconstrictors serotonin and phenylephrine, and inhibited the Ca(2+) signaling evoked by serotonin in vascular smooth muscle cells. Subsequent activity-guided fractionation identified fisetin as an active constituent exerting a Ca(2+) inhibitory effect. Fisetin could inhibit major Ca(2+) mobilization pathways including extracellular Ca(2+) influx mediated by the L-type voltage-gated Ca(2+) channel, Ca(2+) release from the intracellular store and store-operated Ca(2+) entry. In accordance with Ca(2+) inhibitory effect, fisetin attenuated vasoconstriction by serotonin and phenylephrine. These results suggest that the anticontractile effect, which is presumably mediated by inhibition of Ca(2+) signaling, may contribute to the improvement of blood circulation by RVS.

Analyzing isolated blood vessel contraction in multi-well plates.

Organ baths have been successfully used for over a century to study the contractile or relaxation effects of drugs. Indeed, most of our understanding of vascular pharmacology is based on such in vitro studies. However, multiple parallel organ baths that require mechanical transduction consume relatively large amounts of drugs, gases, and buffers, and they take up a considerable bench space. In addition, such experiments have a high demand in terms of cost and animals, and the tissue preparation is labor intensive and slow. For these reasons, organ baths are no longer in the front line of industrial pharmacological research and they have almost disappeared from most academic laboratories. We have developed a very simple system, which can be implemented virtually in any laboratory, for the automatic analyses of rat aorta ring contraction based on optical methods and using multi-well plates. Rat aorta rings (≈0.5 mm wide) were situated in 96-multi-well plates, and the luminal vessel areas were continuously monitored using a USB camera driven by newly developed algorithms. Liquids were handled using multichannel pipettes, although these procedures can be automated for drug screening. The concentration-response curves obtained were similar to those reported in the literature using traditional force transduction techniques on isolated tissues. This system can also be used with other tissue preparations and for simultaneous fluorescence measurements. The new system described here offers a simple, cheap, and reliable alternative to the classic organ bath system.

Brazilin isolated from the heartwood of Caesalpinia sappan L induces endothelium-dependent and -independent relaxation of rat aortic rings

Brazilin is one of the major constituents of Caesalpinia sappan L with various biological activities. This study sought to investigate the vasorelaxant effect of brazilin on isolated rat thoracic aorta and explore the underlying mechanisms. Endothelium-intact and -denuded aortic rings were prepared from rats. The tension of the preparations was recorded isometrically with a force displacement transducer connected to a polygraph. The phosphorylation levels of ERK1/2 and myosin light chain (MLC) were analyzed using Western blotting assay. Application of brazilin (10-100 μmol/L) dose-dependently relaxed the NE- or high K(+)-induced sustained contraction of endothelium-intact aortic rings (the EC50 was 83.51±5.6 and 79.79±4.57 μmol/L, respectively). The vasorelaxant effect of brazilin was significantly attenuated by endothelium removal or by pre-incubation with L-NAME, methylene blue or indomethacin. In addition, pre-incubation with brazilin dose-dependently attenuated the vasoconstriction induced by KCl, NE or Ang II. Pre-incubation with brazilin also markedly suppressed the high K(+)-induced extracellular Ca(2+) influx and NE-induced intracellular Ca(2+) release in endothelium-denuded aortic rings. Pre-incubation with brazilin dose-dependently inhibited the NE-stimulated phosphorylation of ERK1/2 and MLC in both endothelium-intact and -denuded aortic rings. Brazilin induces relaxation in rat aortic rings via both endothelium-dependent and -independent ways as well as inhibiting NE-stimulated phosphorylation of ERK1/2 and MLC. Brazilin also attenuates vasoconstriction via blocking voltage- and receptor-operated Ca(2+) channels.

Mechanisms underlying the antihypertensive effect of Alstonia scholaris

Ethnopharmacological relevanceAlstonia scholaris has a long history of use in the Ayurveda traditional treatment of various ailments including hypertension. We have reported the blood pressure lowering activity of the extract of A. scholaris. The following research aim to delineate the pharmacological mechanism involve in the antihypertensive action. Materials and methodVasorelaxant effect of the n-butanol fraction of A. scholaris (NBF-ASME) was evaluated on rat aorta pre-contracted with phenyelphrine (PE, 1µM). Aortic rings preparation were pre-incubated with various antagonists like 1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ 10μM), methylene blue (MB 10μM), Nω-nitro-L-arginine methyl ester hydrochloride (l-NAME 10μM), atropine (10μM), indomethacin (1μM), ML-9 and various K+ channel blockers such as glibenclamide (10μM) and tetraethyl ammonium (TEA 10μM) for mechanism study. ResultThe results showed that pre-incubation of aortic rings with the extract (0.5, 1 and 2mg/mL) significantly inhibit the contractile response of the rings to phenylephrine-induced contraction (p<0.05–0.001). Removal of endothelium, incubation with l-NAME, indomethacin, atropine and propranolol did not significantly affect the relaxation effect of NBF-ASME. Furthermore, the K+ channel blockers, TEA and glibenclamide showed no inhibitory effect. However, aortic rings pretreated with ODQ and ML-9 showed a significant suppression of the relaxation curve of NBF-ASME (p<0.01–0.001). In Ca2+-free solution, NBF-ASME inhibits the release of intracellular Ca2+ from the sarcoplasmic reticulum. NBF-ASME also inhibits calcium chloride (CaCl2)-induced contraction in endothelium-denuded aortic rings. ConclusionThe results from this study suggests that A. scholaris exerts vasodilation via calcium channels blockade, direct activation of soluble guanylate cyclase and possibly by also inhibiting the formation of inositol 1, 4, 5-triphosphate.

Role of large conductance calcium-activated potassium channels in vascular hyporesponsiveness in rats with obstructive jaundice

Objective To evaluate the role of large conductance calcium-activated potassium (BKCa) channels in vascular hyporesponsiveness in rats with obstructive jaundice. Methods Eighteen male Sprague-Dawley rats, weighing 180-200 g, were randomly divided into 3 groups (n = 6 each) using a random number table: control group (group C), sham operation group (group S), and bile duct ligation group (group BDL). Obstructive jaundice was produced by common bile duct ligation.At 7 days after surgery, blood samples were collected for determination of the levels of serum total bilirubin (TBL), direct bilirubin (DBL), indirect bilirubin (IBL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Thoracic aortic rings were prepared, and the endothelium was removed.The aortic rings were sequentially perfused with different concentrations of norepinephrine (NE) and sodium nitroprusside (SNP), and the maximum amplitude of contraction and dilatation of aortic rings was recorded.The aortic rings were then perfused with BKCa channel blocker Chtx with the final concentration of 10-7 mol/L, followed by perfusion with different concentrations of NE and SNP again, and the maximum amplitude of contraction and dilatation of aortic rings was recorded under each concentration.The percentage of maximum contraction and dilatation (maximum amplitude after Chtx administration÷maximum amplitude before Chtx administration×100%) was calculated. Results Compared with C and S groups, the levels of TBL, DBL, IBL, ALT and AST in serum were significantly increased, the maximum amplitude of NE-induced contraction of aortic rings was decreased, and the percentage of the maximum NE-induced dilatation of aortic rings was increased, the maximum amplitude of SNP-induced contraction of aortic rings was increased, and the percentage of the maximum SNP-induced dilatation of aortic rings was decreased in group BDL. Conclusion Excessive opening of BKCa channels may be involved in the mechanism of vascular hyporesponsiveness in rats with obstructive jaundice. Key words: Jaundice, obstructive; Large-conductance calcium-activated potassium channels; Aorta, thoracic

Abstract 642: Wy14643 and Fenofibrate Reduce Acetylcholine-induced Contractions in Spontaneously Hypertensive Rat Aortae

Introduction and hypothesis: Hypertension is associated with endothelial dysfunction, which is defined as an imbalanced release of endothelium-derived relaxing factors (EDRF) and endothelium-derived contracting factors (EDCF). Oxidative stress contributes to endothelial dysfunction by increasing the production of EDCF. Research studies have demonstrated that peroxisome proliferator-activated receptor alpha agonists, Wy14643 (WY) and fenofibrate (FF), improve endothelial function. Therefore, we assessed the hypothesis that WY and FF inhibited acetylcholine-induced endothelium-dependent contractions through reduction of oxidative stress in aortae of spontaneously hypertensive rats (SHR). Method: Thoracic aortic rings, with and without endothelium, were isolated from 40-44 weeks old male SHR and their normotensive counterparts, Wistar Kyoto rats (WKY). They were suspended in organ chambers for isometric tension recording. Responses to the endothelium-dependent vasoactive agent, acetylcholine (10 -8 - 10 -4 M), and to the oxidizing agent, hydrogen peroxide (10 -8 - 10 -4 M), were examined in the presence of N ω -nitro-L-arginine methyl ester (L-NAME, 3*10 -4 M; nitric oxide synthase inhibitor). Results: Acetylcholine caused significantly greater contractions in aortic rings, with endothelium, of SHR than in those of WKY. The contraction was significantly inhibited by the combination of Tiron (10 -3 M; intracellular superoxide anion scavenger) and DETCA (10 -4 M; superoxide dismutase inhibitor), suggesting that the reactive oxygen species, hydrogen peroxide, was involved in vascular dysfunction in hypertension. Both WY (10 -5 M) and FF (10 -5 M) significantly inhibited contractions to acetylcholine in SHR aorta, suggesting that they may reduce the amount of hydrogen peroxide or inhibit its downstream signaling. Moreover, they inhibited contractions induced by hydrogen peroxide in SHR aortic rings with, but not in those without, endothelium, indicating that their inhibitory effect was endothelium-dependent. Conclusion: The peroxisome proliferator-activated receptor alpha agonists act on the endothelium to reduce acetylcholine-induced contractions that are mediated by hydrogen peroxide in aortae of hypertensive rats.

Vascular Reactivity of Rat, Guinea‐pig, Rabbit and Human Vessels to Bufadienolides and Cardenolides

Cardenolides and bufodienolides are Na+-K+-ATPase (NKA) inhibitors. The aim of the present study was to evaluate the response to such compounds of vessels from different species in order to compare potency and efficacy. The experimental protocols were approved by the Animal Care and Ethics Committee of the Ceara State University. The aortic rings and mesenteric artery for isometric recordings of contractions in response to cumulative addition of OUA, BUF, DIG (0.1-100 µM). The contractions were expressed as percentage of the maximal contraction elicited by phenylephrine. Ouabain, up to 100 µM, promoted no contraction in rat aorta but promoted a slow onset contraction that attained a maximum of 71.5±14.8% and 101.2±8.5% in guinea-pig and rabbit tissue, respectively. Bufalin promoted slow onset contractions after 30 and 100 µM that reached a maximum of 20.6 ± 5.2%,55.0±10.5% and 94±19.8% in tissues gathered from rats, guinea-pigs and rabbits, respectively. On the other hand, digoxin did not elicit contraction in rabbit or rat aortic rings but elicited a similar slow onset that attained its maximum after more than 50 minutes and reached 79.7±15.6% in guinea-pig tissues. In human aortic rings in response to 100 µM bufalin or ouabain reached a maximum of 96.7±24.4% or 140±26.7%, respectively. In the 2nd branch of the mesenteric artery, BUF promoted a slow contractile response (46 % ± 9.6 %) only at a dose of 100 µM.Pretreatment with guanethidine (GUA + BUF) significantly attenuated BUF vasoconstriction in aortic rings (39.7% ± 8.4) and prazosin (1µM) completely blocked the contraction, suggesting and indirect noradrenergic activation.

Discovery of phenoxybutanoic acid derivatives as potent endothelin antagonists with antihypertensive activity

A series of phenoxybutanoic acid derivatives were synthesized and tested for their antagonistic activity on the contraction of the rat thoracic aortic ring induced by endothelin-1. Preliminary screening results showed that 6e and 6g with benzoheterocycles demonstrated significant antagonistic activities when compared to the reference compound BQ123. The results from additional assays for the binding affinity and selectivity for endothelin receptors showed that 6e was a selective ETA antagonist with a nanomolar IC50. Moreover, 6e was effective in relieving hypoxia-induced pulmonary arterial hypertension and right ventricular weight ratio. Therefore, 6e may have potential for further development as a therapeutic agent for the treatment of cardiovascular diseases.

Cardioprotective effects of voluntary exercise in a rat model: role of matrix metalloproteinase-2.

Background. Regular exercise at moderate intensity reduces cardiovascular risks. Matrix metalloproteinases (MMPs) play a major role in cardiac remodeling, facilitating physiological adaptation to exercise. The aim of this study was to examine the influence of voluntary physical exercise on the MMP-2 enzyme activity and to investigate the cardiac performance by measurement of angina susceptibility of the heart, the basal blood pressure, the surviving aorta ring contraction, and the cardiac infarct size after I/R-induced injury. Methods. Male Wistar rats were divided into control and exercising groups. After a 6-week period, the serum level of MMP-2, basal blood pressure, cardiac angina susceptibility (the ST segment depression provoked by epinephrine and 30 s later phentolamine), AVP-induced heart perfusion and aorta ring contraction, infarct size following 30 min ischemia and 120 min reperfusion, and coronary effluent MMP-2 activity were measured. Results. Voluntary wheel-running exercise decreased both the sera (64 kDa and 72 kDa) and the coronary effluent (64 kDa) MMP-2 level, reduced the development of ST depression, improved the isolated heart perfusion, and decreased the ratio of infarct size. Conclusion. 6 weeks of voluntary exercise training preserved the heart against cardiac injury. This protective mechanism might be associated with the decreased activity of MMP-2.

Glutathione Supplementation Attenuates Oxidative Stress and Improves Vascular Hyporesponsiveness in Experimental Obstructive Jaundice.

We investigated the protective effects and mechanism of glutathione (GSH) on vascular hyporesponsiveness induced by bile duct ligation (BDL) in a rat model. Seventy-two male Sprague-Dawley rats were randomly divided into four groups: a NS group, a GSH group, a BDL + NS group, and a BDL + GSH group. GSH was administrated into rats in the GSH and BDL + GSH groups by gastric gavage. An equal volume of normal saline was, respectively, given in the NS group and BDL + NS group. Blood was gathered for serological determination and thoracic aorta rings were isolated for measurement of isometric tension. Obstructive jaundice led to a significant increase in the serum total bilirubin, AST, and ALT levels. The proinflammatory cytokines levels (TNF-α and IL-1β), concentration of NO, and oxidative stress markers (MDA and 3-NT) were increased as well. All of those were reduced by the treatment of GSH. Meanwhile, contraction of aorta rings to NA and vasorelaxation to ACh or SNP in the BDL group rats were markedly decreased, while GSH administration reversed this change. Our findings suggested that GSH supplementation attenuated overexpressed ONOO(−) from the reaction of excessive NO with O2 ∙- and protected against obstructive jaundice-induced vascular hyporesponsiveness in rats.

Angiotensin II Type I and Prostaglandin F2α Receptors Cooperatively Modulate Signaling in Vascular Smooth Muscle Cells

The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gαq signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure.

Vasorelaxation induced by methyl cinnamate, the major constituent of the essential oil of Ocimum micranthum, in rat isolated aorta.

The aim of the present study was to investigate the vascular effects of the E-isomer of methyl cinnamate (E-MC) in rat isolated aortic rings and the putative mechanisms underlying these effects. At 1-3000μmol/L, E-MC concentration-dependently relaxed endothelium-intact aortic preparations that had been precontracted with phenylephrine (PHE; 1μmol/L), with an IC50 value (geometric mean) of 877.6μmol/L (95% confidence interval (CI) 784.1-982.2μmol/L). These vasorelaxant effects of E-MC remained unchanged after removal of the vascular endothelium (IC50 725.5μmol/L; 95% CI 546.4-963.6μmol/L) and pretreatment with 100μmol/L N(G) -nitro-l-arginine methyl ester (IC50 749.0μmol/L; 95% CI 557.8-1005.7μmol/L) or 10μmol/L 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (IC50 837.2μmol/L; 95% CI 511.4-1370.5μmol/L). Over the concentration range 1-3000μmol/L, E-MC relaxed K(+) -induced contractions in mesenteric artery preparations (IC50 314.5μmol/L; 95% CI 141.9-697.0μmol/L) with greater potency than in aortic preparations (IC50 1144.7μmol/L; 95% CI 823.2-1591.9μmol/L). In the presence of a saturating contractile concentration of K(+) (150mmol/L) in Ca(2+) -containing medium combined with 3μmol/L PHE, 1000μmol/L E-MC only partially reversed the contractile response. In contrast, under similar conditions, E-MC nearly fully relaxed PHE-induced contractions in aortic rings in a Ba(2+) -containing medium. In preparations that were maintained under Ca(2+) -free conditions, 600 and 1000μmol/L E-MC significantly reduced the contractions induced by exogenous Ca(2+) or Ba(2+) in KCl-precontracted preparations, but not in PHE-precontracted preparations (in the presence of 1μmol/L verapamil). In addition, E-MC (1-3000μmol/L) concentration-dependently relaxed the contractions induced by 2mmol/L sodium orthovanadate. Based on these observations, E-MC-induced endothelium-independent vasorelaxant effects appear to be preferentially mediated by inhibition of plasmalemmal Ca(2+) influx through voltage-dependent Ca(2+) channels. However, the involvement of a myogenic mechanism in the effects of E-MC is also possible.

ACUTE KIDNEY INJURY DECREASED ENDOGENOUS HYDROGEN SULFIDE PRODUCTION AND INCREASED CYTOKINE EXPRESSION: IMPACT ON CARDIOVASCULAR SYSTEM

BACKGROUND: In some pathologic circumstances (e.g., septicemia) nitric oxide (NO) synthesis is higher and it is implicated in lowering of the blood pressure that usually is less responsive to conventional therapy with adrenaline and vasopressin. Thus, new specific therapeutic approaches using high affinity NO scavengers are of great relevance. Accordingly, some new ruthenium compounds with this putative property were synthesized. The aim of this work was to evaluate the NO scavenging activity of those compounds. METHODS: Aorta rings obtained from the thoracic aorta of adult male Wistar rats were suspended under 60% of maximal tension in an organ bath, containing 15 mL of Krebs-Henseleit solution (36.5 0.1oC, pH 7.2-7.4, gassed with 95% O2:5% CO2). After 60 min stabilization period, maximum contraction of aortic rings was obtained in response to noradrenaline. After that, acetylcholine was added in order to obtain maximal relaxation. Cumulative concentration-response curves were obtained to the compounds: trans-[Ru(NH3)4(SO4)(4-picoline)](BF4) (Ru-pic); trans-[Ru(NH3)4 (SO4) (nicotinamida)](BF4) (Ru-ina); trans-[Ru(NH3)4(SO4) (isonicotina mide)] (BF4) (Ru-isn); [Ru(NH3)5Cl]Cl2 (Ru-Cl); [Ru(edta)Cl] (RuEDTA); cis-[Ru(NH3)4oxalato](TFMS)3 (Ru-cis-Ox); cis[Ru(NH3)4(H2O)2 (TFMS)3 (Ru-cis-aquo) and trans [Ru(NH3)5(H2O)(TFMS)3 (Ru-trans-aquo). The same procedure was performed in the presence of L-NAME, an inhibitor of nitric oxide synthase. Control rings were not exposed to any ruthenium compound. RESULTS: The mean sem of the tension developed by the tissues following exposition to noradrenaline was 1.94 0.17 gf. Acetylcholine reduced the developed tension to 0.17 0.04 gf. In the presence of the all Ru-compounds, the developed tensions (gf) were: 1.68 0.19 (Ru-pic; n1⁄45); 1.43 0.15 (Ru-isn; n1⁄45); 1.47 0.16 (Ru-ina; n1⁄45); 1.74 0.13 (Ru-EDTA; n1⁄45); 2.93 0.24 (Ru-Cl; n1⁄45); 2.85 0.07 (Ru-cis-Ox; n1⁄45); 3.08 0.15 (Ru-cis-aquo; n1⁄45); 3.12 0.12 (Ru-trans-aquo; n1⁄45); [p<0.05 compared to control, Student t test]. All the analyzed compounds were able to cancel the effect of acetylcholine and had similar potencies (pD2; mean sem): 5.60 0.18 (Ru-pic); 5.34 0.25 (Ru-isn); 5.40 0.27 (Ru-ina); 5.48 0.23 (Ru-Cl); 6.45 0.39 (Ru-EDTA); 5.19 0.30 (Ru-cis-Ox); 5.59 0.24 (Ru-cis-aquo); 5.03 0.37 (Ru-trans-aquo). None of the effects of acetylcholine or compounds were observed when they were tested in the presence of L-NAME. CONCLUSION: The data suggested that all compounds were able to scavenge NO in rat aortic rings and to restore the vascular tone.